Purified thermostable enzyme

ABSTRACT

Recombinant DNA vectors that encode a thermostable DNA polymerase are useful in the recombinant production of thermostable DNA polymerase. The recombinant thermostable polymerase is preferred for use in the production of DNA in a polymerase chain reaction. Especially useful vectors encode the  DIFFERENCE 94,000 dalton thermostable DNA polymerase from thermus aquaticus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of now abandoned Ser. No. 143,441,filed Jan. 12, 1988, which is a continuation in part of Ser. No.063,509, filed June 17, 1987, which issued as U.S. Pat. No. 4,889,818,and which is a continuation in part of Ser. No. 899,241, filed Aug. 22,1986, and now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a purified thermostable enzyme. In oneembodiment the enzyme is DNA polymerase purified from Thermus aquaticusand has a molecular weight of about 86,000-95,000. In another embodimentthe enzyme is DNA polymerase produced by recombinant means.

2. Background Art

Extensive research has been conducted on the isolation of DNApolymerases from mesophilic microorganisms such as E. coli. See, forexample, Bessman et al., J. Biol. Chem. (1957) 233: 171-177 and Buttinand Kornberg (1966) J. Biol. Chem. 241: 5419-5427.

In contrast, relatively little investigation has been made on theisolation and purification of DNA polymerases from thermophiles, such asThermus aquaticus. Kaledin et al., Biokymiya (1980) 45: 644-651discloses a six-step isolation and purification procedure of DNApolymerase from cells of T. aquaticus YT1 strain. These steps involveisolation of crude extract, DEAE-cellulose chromatography, fractionationon hydroxyapatite, fractionation on DEAE-cellulose, and chromatographyon single-strand DNA-cellulose. The pools from each stage were notscreened for contaminating endo- and exonuclease(s). The molecularweight of the purified enzyme is reported as 62,000 daltons permonomeric unit.

A second purification scheme for a polymerase from T. aquaticus isdescribed by A. Chien et al., J. Bacteriol. (1976) 127: 1550-1557. Inthis process, the crude extract is applied to a DEAE-Sephadex column.The dialyzed pooled fractions are then subjected to treatment on aphosphocellulose column. The pooled fractions are dialyzed and bovineserum albumin (BSA) is added to prevent loss of polymerase activity. Theresulting mixture is loaded on a DNA-cellulose column. The pooledmaterial from the column is dialyzed and analyzed by gel filtration tohave a molecular weight of about 63,000 daltons, and, by sucrosegradient centrifugation of about 68,000 daltons.

The use of a thermostable enzyme to amplify existing nucleic acidsequences in amounts that are large compared to the amount initiallypresent has been suggested in U.S. Pat. No. 4,683,195. Primers,nucleotide triphosphates, and a polymerase are used in the process,which involves denaturation, synthesis of template strands andhybridization. The extension product of each primer becomes a templatefor the production of the desired nucleic acid sequence. The patentdiscloses that if the polymerase employed is a thermostable enzyme, itneed not be added after every denaturation step, because the heat willnot destroy its activity. No other advantages or details are provided onthe use of a purified thermostable DNA polymerase. Furthermore, NewEngland Biolabs had marketed a polymerase from T. aquaticus, but wasunaware that the polymeras activity decreased substantially with time ina storage buffer not containing non-ionic detergents.

Accordingly, there is a desire in the art to produce a purified, stablethermostable enzyme that may be used to improve the nucleic acidamplification process described above.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a purified thermostableenzyme that catalyzes combination of nucleotide triphosphates to form anucleic acid strand complementary to a nucleic acid template strand.Preferably the purified enzyme is DNA polymerase from Thermus aquaticusand has a molecular weight of about 86,000-95,000 daltons. This purifiedmaterial may be used in a temperature-cycling amplification reactionwherein nucleic acid sequences are produced from a given nucleic acidsequence in amounts that are large compared to the amount initiallypresent so that they can be manipulated and/or analyzed easily.

The gene encoding the DNA polymerase enzyme from Thermus aquaticus hasalso been identified and cloned and provides yet another means toprepare the thermostable enzyme of the present invention. In addition tothe gene encoding the approximately 86,000-95,000 dalton enzyme, genederivatives encoding DNA polymerase activity are also presented.

The invention also encompasses a stable enzyme composition comprising apurified, thermostable enzyme as described above in a buffer containingone or more non-ionic polymeric detergents.

Finally, the invention provides a method of purification for thethermostable polymerase of the invention which comprises treating anaqueous mixture containing the thermostable polymerase with ahydrophobic interaction chromatographic support under conditions whichpromote hydrophobic interactions and eluting the bound thermostablepolymerase from said support with a solvent which attenuates hydrophobicinteractions.

The purified enzyme, as well as the enzymes produced by recombinant DNAtechniques, provide much more specificity than the Klenow fragment,which is not thermostable, when used in the temperature-cyclingamplification reaction. In addition, the purified enzyme and therecombinantly produced enzymes exhibit the appropriate activity expectedwhen TTP or other nucleotide triphosphates are not present in theincubation mixture with the DNA template. Also, the enzymes herein havea broader pH profile than that of the thermostable enzyme from Thermusaquaticus described in the literature, with more than 50% of theactivity at pH 6.4 as at pH 8.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the DNA sequence and the predicted amino acid sequence for Taqpolymerase. The amino acid sequence corresponding to the deduced primarytranslation product is numbered 1-832.

FIG. 2 is a restriction site map of plasmid pFC83 that contains the ˜4.5kb HindIII T. aquaticus DNA insert subcloned into plasmid BSM13+.

FIG. 3 is a restriction site map of plasmid pFC85 that contains the˜2.68 kb HindIII to Asp718 T. aquaticus DNA insert subcloned intoplasmid BSM13+.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, "cell", "cell line", and "cell culture" can be usedinterchangeably and all such designations include progeny. Thus, thewords "transformants" or "transformed cells" includes the primarysubject cell and cultures derived therefrom without regard for thenumber of transfers. It is also understood that all progeny may not beprecisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same functionality as screenedfor in the originally transformed cell are included.

The term "control sequences" refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for procaryotes, forexample, include a promoter, optionally an operator sequence, a ribosomebinding site, and possibly, other as yet poorly understood sequences.Eucaryotic cells are known to utilize promoters, polyadenylationsignals, and enhancers.

The term "expression system" refers to DNA sequences containing adesired coding sequence and control sequences in operable linkage, sothat hosts transformed with these sequences are capable of producing theencoded proteins. In order to effect transformation, the expressionsystem may be included on a vector; however, the relevant DNA may thenalso be integrated into the host chromosome.

The term "gene" as used herein refers to a DNA sequence that encodes arecoverable bioactive polypeptide or precursor. The polypeptide can beencoded by a full-length gene sequence or any portion of the codingsequence so long as the enzymatic activity is retained.

In one embodiment of the invention, the DNA sequence encoding afull-length thermostable DNA polymerase of Thermus aquaticus (Taq) isprovided. FIG. 1 shows this DNA sequence and the deduced amino acidsequence. For convenience, the amino acid sequence of this Taqpolymerase will be used as a reference and other forms of thethermostable enzyme will be designated by referring to the sequenceshown in FIG. 1. Since the N-terminal methionine may or may not bepresent, both forms are included in all cases wherein the thermostableenzyme is produced in bacteria.

"Operably linked" refers to juxtaposition such that the normal functionof the components can be performed. Thus, a coding sequence "operablylinked" to control sequences refers to a configuration wherein thecoding sequences can be expressed under the control of the controlsequences.

The term "mixture" as it relates to mixtures containing Taq polymeraserefers to a collection of materials which includes Taq polymerase butwhich also includes alternative proteins. If the Taq polymerase isderived from recombinant host cells, the other proteins will ordinarilybe those associated with the host. Where the host is bacterial, thecontaminating proteins will, of course, be bacterial proteins.

"Non-ionic polymeric detergents" refers to surface-active agents thathave no ionic charge and that are characterized, for purposes of thisinvention, by their ability to stabilize the enzyme herein at a pH rangeof from about 3.5 to about 9.5, preferably from 4 to 8.5.

The term "oligonucleotide" as used herein is defined as a moleculecomprised of two or more deoxyribonucleotides or ribonucleotides,preferably more than three. Its exact size will depend on many factors,which in turn depend on the ultimate function or use of theoligonucleotide. The oligonucleotide may be derived synthetically or bycloning.

The term "primer" as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinitiated, i.e., in the presence of four different nucleotidetriphosphates and thermostable enzyme in an appropriate buffer ("buffer"includes pH, ionic strength, cofactors, etc.) and at a suitabletemperature. For Taq polymerase the buffer herein preferably contains1.5-2 mM of a magnesium salt, preferably MgCl₂, 150-200 μM of eachnucleotide, and 1 μM of each primer, along with preferably 50 mM KCl, 10mM Tris buffer, pH 8-8.4, and 100 μg/ml gelatin.

The primer is preferably single-stranded for maximum efficiency inamplification, but may alternatively be double-stranded. Ifdouble-stranded, the primer is first treated to separate its strandsbefore being used to prepare extension products. Preferably, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of thethermostable enzyme. The exact lengths of the primers will depend onmany factors, including temperature, source of primer and use of themethod. For example, depending on the complexity of the target sequence,the oligonucleotide primer typically contains 15-25 nucleotides,although it may contain more or fewer nucleotides. Short primermolecules generally require cooler temperatures to form sufficientlystable hybrid complexes with template.

The primers herein are selected to be "substantially" complementary tothe different strands of each specific sequence to be amplified. Thismeans that the primers must be sufficiently complementary to hybridizewith their respective strands. Therefore, the primer sequence need notreflect the exact sequence of the template. For example, anon-complementary nucleotide fragment may be attached to the 5' end ofthe primer, with the remainder of the primer sequence beingcomplementary to the strand. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementarity with the sequence of thestrand to be amplified to hybridize therewith and thereby form atemplate for synthesis of the extension product of the other primer.However, for detection purposes, particulary using labeledsequence-specific probes, the primers typically have exactcomplementarity to obtain the best results.

As used herein, the terms "restriction endonucleases" and "restrictionenzymes" refer to bacterial enzymes each of which cut double-strandedDNA at or near a specific nucleotide sequence.

As used herein, the term "thermostable enzyme" refers to an enzyme whichis stable to heat and is heat resistant and catalyzes (facilitates)combination of the nucleotides in the proper manner to form the primerextension products that are complementary to each nucleic acid strand.Generally, the synthesis will be initiated at the 3' end of each primerand will proceed in the 5' direction along the template strand, untilsynthesis terminates, producing molecules of different lengths. Theremay be a thermostable enzyme, however, which initiates synthesis at the5' end and proceeds in the other direction, using the same process asdescribed above.

The thermostable enzyme herein must satisfy a single criterion to beeffective for the amplification reaction, i.e., the enzyme must notbecome irreversibly denatured (inactivated) when subjected to theelevated temperatures for the time necessary to effect denaturation ofdouble-stranded nucleic acids. Irreversible denaturation for purposesherein refers to permanent and complete loss of enzymatic activity. Theheating conditions necessary for nucleic acid denaturation will depend,e.g., on the buffer salt concentration and composition and the lengthand nucleotide composition of the nucleic acids being denatured, buttypically range from about 90° to about 105° C. for a time dependingmainly on the temperature and the nucleic acid length, typically about0.5 to four minutes. Higher temperatures may be tolerated as the buffersalt concentration and/or GC composition of the nucleic acid isincreased. Preferably, the enzyme will not become irreversibly denaturedat about 90°-100° C.

The thermostable enzyme herein preferably has an optimum temperature atwhich it functions that is higher than about 40° C., which is thetemperature below which hybridization of primer to template is promoted,although, depending on (1) salt concentration and composition and (2)composition and length of primer, hybridization can occur at highertemperature (e.g., 45°-70° C.). The higher the temperature optimum forthe enzyme, the greater the specificity and/or selectivity of theprimer-directed extension process. However, enzymes that are activebelow 40° C., e.g., at 37° C., are also within the scope of thisinvention provided they are heat-stable. Preferably, the optimumtemperature ranges from about 50° to 90° C., more preferably 60°-80° C.

The thermostable enzyme herein may be obtained from any source and maybe a native or recombinant protein. Examples of enzymes that have beenreported in the literature as being resistant to heat includeheat-stable polymerases, such as, e.g., polymerases extracted from thethermophilic bacteria Thermus flavus, Thermus ruber, Thermusthermophilus, Bacillus stearothermophilus (which has a somewhat lowertemperature optimum than the others listed), Thermus aquaticus, Thermuslacteus, Thermus rubens, and Methanothermus fervidus. In addition,thermostable polymerases isolated from the thermophilic archaebacteriainclude, for example, Sulfolobus solfataricus, Sulfolobusacidocaldarius, Thermoplasma acidophilum, Methanobacteriumthermoautotrophicum, and Desulfurococcus mobilis.

The thermostable enzyme of the invention has the amino acid sequencepresented in FIG. 1. In addition, any thermostable polymerase containingat least 50% homology to any contiguous stretch of nine or more aminoacids presented therein is also intended to be within the scope of theinvention. This homology can be determined using commercially availabledata banks such as the European Molecular Biology Laboratory (EMBL) orGenbank. Moreover, as new thermostable polymerases are identified,specific regions of homology between the newly identified sequences andthe Taq polymerase sequence may be determined using, for example, theSequence Analysis Software Package of the Genetics Computer Group of theUniversity of Wisconsin. Specific regions of homology include thefollowing sequences (numbered according to the numbering of amino acidsin FIG. 1): residues 190-204, 262-270, 569-587, 718-732, 743-759, and778-790.

The preferred thermostable enzyme herein is a DNA polymerase isolatedfrom Thermus aquaticus. Various strains thereof are available from theAmerican Type Culture Collection, Rockville, Md., and are described byT. D. Brock, J. Bact. (1969) 98: 289-297, and by T. Oshima, Arch.Microbiol. (1978) 117: 189-196. One of these preferred strains is strainYT-1.

For recovering the native protein the cells are grown using any suitabletechnique. One such technique is described by Kaledin et al., Biokhimiya(1980), supra, the disclosure of which is incorporated herein byreference. Briefly, the cells are grown on a medium, in one liter, ofnitrilotriacetic acid (100 mg), tryptone (3 g), yeast extract (3 g),succinic acid (5 g), sodium sulfite (50 mg), riboflavin (1 mg), K₂ HPO₄(522 mg), MgSO₄ (480 mg), CaCl₂ (222 mg), NaCl (20 mg), and traceelements. The pH of the medium is adjusted to 8.0±0.2 with KOH. Theyield is increased up to 20 grams of cells/liter if cultivated withvigorous aeration at a temperature of 70° C. Cells in the latelogarithmic growth stage (determined by absorbance at 550 nm) arecollected by centrifugation, washed with a buffer and stored frozen at-20° C.

In another method for growing the cells, described in Chien et al., J.Bacteriol. (1976), supra, the disclosure of which is incorporated hereinby reference, a defined mineral salts medium containing 0.3% glutamicacid supplemented with 0.1 mg/l biotin, 0.1 mg/l thiamine, and 0.05 mg/lnicotinic acid is employed. The salts include nitrilotriacetic acid,CaSO₄, MgSO₄, NaCl, KNO₃, NaNO₃, ZnSO₄, H₃ BO₃, CuSO₄, NaMoO₄, CoCl₂,FeCl₃, MnSO₄, and Na₂ HPO₄. The pH of the medium is adjusted to 8.0 withNaOH.

In the Chien et al. technique, the cells are grown initially at 75° C.in a water bath shaker. On reaching a certain density, 1 liter of thesecells is transferred to 16-liter carboys which are placed in hot-airincubators. Sterile air is bubbled through the cultures and thetemperature maintained at 75° C. The cells are allowed to grow for 20hours before being collected by centrifuge.

After cell growth, the isolation and purification of the enzyme takeplace in six stages, each of which is carried out at a temperature belowroom temperature, preferably about 4° C.

In the first stage or step, the cells, if frozen, are thawed,disintegrated by ultrasound, suspended in a buffer at about pH 7.5, andcentrifuged.

In the second stage, the supernatant is collected and then fractionatedby adding a salt such as dry ammonium sulfate. The appropriate fraction(typically 45-75% of saturation) is collected, dissolved in a 0.2Mpotassium phosphate buffer preferably at pH 6.5, and dialyzed againstthe same buffer.

The third step removes nucleic acids and some protein. The fraction fromthe second stage is applied to a DEAE-cellulose column equilibrated withthe same buffer as used above. Then the column is washed with the samebuffer and the flow-through protein-containing fractions, determined byabsorbance at 280 nm, are collected and dialyzed against a 10 mMpotassium phosphate buffer, preferably with the same ingredients as thefirst buffer, but at a pH of 7.5.

In the fourth step, the fraction so collected is applied to ahydroxyapatite column equilibrated with the buffer used for dialysis inthe third step. The column is then washed and the enzyme eluted with alinear gradient of a buffer such as 0.01M to 0.5M potassium phosphatebuffer at pH 7.5 containing 10 mM 2-mercaptoethanol and 5% glycerine.The pooled fractions containing thermostable enzyme (e.g., DNApolymerase) activity are dialyzed against the same buffer used fordialysis in the third step.

In the fifth stage, the dialyzed fraction is applied to a DEAE-cellulosecolumn, equilibrated with the buffer used for dialysis in the thirdstep. The column is then washed and the enzyme eluted with a lineargradient of a buffer such as 0.01 to 0.6M KCl in the buffer used fordialysis in the third step. Fractions with thermostable enzyme activityare then tested for contaminating deoxyribonucleases (endo- andexonucleases) using any suitable procedure. For example, theendonuclease activity may be determined electrophoretically from thechange in molecular weight of phage λ DNA or supercoiled plasmid DNAafter incubation with an excess of DNA polymerase. Similarly,exonuclease activity may be determined electrophoretically from thechange in molecular weight of DNA after treatment with a restrictionenzyme that cleaves at several sites.

The fractions determined to have no deoxyribonuclease activity arepooled and dialyzed against the same buffer used in the third step.

In the sixth step, the pooled fractions are placed on a phosphocellulosecolumn with a set bed volume. The column is washed and the enzyme elutedwith a linear gradient of a buffer such as 0.01 to 0.4M KCl in apotassium phosphate buffer at pH 7.5. The pooled fractions havingthermostable polymerase activity and no deoxyribonuclease activity aredialyzed against a buffer at pH 8.0.

The molecular weight of the dialyzed product may be determined by anytechnique, for example, by SDS-PAGE analysis using protein molecularweight markers. The molecular weight of one of the preferred enzymesherein, the DNA polymerase purified from Thermus aquaticus, isdetermined by the above method to be about 86,000-90,000 daltons. Themolecular weight of this same DNA polymerase as determined by thepredicted amino acid sequence is calculated to be approximately 94,000daltons. Thus, the molecular weight of the full length DNA polymerase isdependent upon the method employed to determine this number and fallswithin the range of 86,000-95,000 daltons.

The thermostable enzyme of this invention may also be produced byrecombinant DNA techniques, as the gene encoding this enzyme has beencloned from Thermus aquaticus genomic DNA. The complete coding sequencefor the Thermus aquaticus (Taq) polymerase can be derived frombacteriophage CH35: Taq#4-2 on an approximately 3.5 kilobase (kb)BglII-Asp718 (partial) restriction fragment contained within an ˜18 kbgenomic DNA insert fragment. This bacteriophage was deposited with theAmerican Type Culture Collection (ATCC) on May 29, 1987 and hasaccession no. 40,336. Alternatively, the gene can be constructed byligating an ˜730 base pair (bp) BglII-HindIII restriction fragmentisolated from plasmid pFC83 (ATCC 67,422 deposited May 29, 1987) to an˜2.68 kb HindIII-Asp718 restriction fragment isolated from plasmid pFC85(ATCC 67,421 deposited May 29, 1987). The pFC83 restriction fragmentcomprises the amino-terminus of the Taq polymerase gene while therestriction fragment from pFC85 comprises the carboxy-terminus. Thus,ligation of these two fragments into a correspondingly digested vectorwith appropriate control sequences will result in the translation of afull-length Taq polymerase.

As stated previously, the DNA and deduced amino acid sequence of apreferred thermostable enzyme is provided in FIG. 1. In addition to theN-terminal deletion described supra, it has also been found that theentire coding sequence of the Taq polymerase gene is not required torecover a biologically active gene product with DNA polymerase activity.Amino-terminal deletions wherein approximately one-third of the codingsequence is absent has resulted in producing a gene product that isquite active in polymerase assays.

In addition to the N-terminal deletions, individual amino acid residuesin the peptide chain comprising Taq polymerase may be modified byoxidation, reduction, or other derivatization, and the protein may becleaved to obtain fragments that retain activity. Such alterations thatdo not destroy activity do not remove the protein from the definition,and are specifically included.

Thus, modifications to the primary structure itself by deletion,addition, or alteration of the amino acids incorporated into thesequence during translation can be made without destroying the hightemperature DNA polymerase activity of the protein. Such substitutionsor other alterations result in proteins having an amino acid sequenceencoded by DNA falling within the contemplated scope of the presentinvention.

Polyclonal antiserum from rabbits immunized with the purified86,000-95,000 dalton polymerase of this invention was used to probe aThermus aquaticus partial genomic expression library to obtain theappropriate coding sequence as described below. The cloned genomicsequence can be expressed as a fusion polypeptide, expressed directlyusing its own control sequences, or expressed by constructions usingcontrol sequences appropriate to the particular host used for expressionof the enzyme.

Of course, the availability of DNA encoding these sequences provides theopportunity to modify the codon sequence so as to generate mutein(mutant protein) forms also having DNA polymerase activity.

Thus, these tools can provide the complete coding sequence for Taq DNApolymerase from which expression vectors applicable to a variety of hostsystems can be constructed and the coding sequence expressed. Portionsof the Taq polymerase-encoding sequence are useful as probes to retrieveother thermostable polymerase-encoding sequences in a variety ofspecies. Accordingly, portions of the genomic DNA encoding at least fourto six amino acids can be replicated in E. coli and the denatured formsused as probes or oligodeoxyribonucleotide probes can be synthesizedwhich encode at least four to six amino acids and used to retrieveadditional DNAs encoding a thermostable polymerase. Because there maynot be a precisely exact match between the nucleotide sequence in theThermus aquaticus form and that in the corresponding portion of otherspecies, oligomers containing approximately 12-18 nucleotides (encodingthe four to six amino acid stretch) are probably necessary to obtainhybridization under conditions of sufficient stringency to eliminatefalse positives. The sequences encoding six amino acids would supplyinformation sufficient for such probes.

SUITABLE HOSTS, CONTROL SYSTEMS AND METHODS

In general terms, the production of a recombinant form of Taq polymerasetypically involves the following:

First, a DNA is obtained that encodes the mature (used here to includeall muteins) enzyme or a fusion of the Taq polymerase to an additionalsequence that does not destroy its activity or to an additional sequencecleavable under controlled conditions (such as treatment with peptidase)to give an active protein. If the sequence is uninterrupted by intronsit is suitable for expression in any host. This sequence should be in anexcisable and recoverable form.

The excised or recovered coding sequence is then preferably placed inoperable linkage with suitable control sequences in a replicableexpression vector. The vector is used to transform a suitable host andthe transformed host cultured under favorable conditions to effect theproduction of the recombinant Taq polymerase. Optionally the Taqpolymerase is isolated from the medium or from the cells; recovery andpurification of the protein may not be necessary in some instances,where some impurities may be tolerated.

Each of the foregoing steps can be done in a variety of ways. Forexample, the desired coding sequences may be obtained from genomicfragments and used directly in appropriate hosts. The constructions forexpression vectors operable in a variety of hosts are made usingappropriate replicons and control sequences, as set forth below.Suitable restriction sites can, if not normally available, be added tothe ends of the coding sequence so as to provide an excisable gene toinsert into these vectors.

The control sequences, expression vectors, and transformation methodsare dependent on the type of host cell used to express the gene.Generally, procaryotic, yeast, insect or mammalian cells are presentlyuseful as hosts. Procaryotic hosts are in general the most efficient andconvenient for the production of recombinant proteins and thereforepreferred for the expression of Taq polymerase.

In the particular case of Taq polymerase, evidence indicates thatconsiderable deletion at the N-terminus of the protein may occur underboth recombinant and native conditions, and that the DNA polymeraseactivity of the protein is still retained. It appears that the nativeproteins previously isolated may be the result of proteolyticdegradation, and not translation of a truncated gene. The muteinproduced from the truncated gene of plasmid pFC85 is, however, fullyactive in assays for DNA polymerase, as is that produced from DNAencoding the full-length sequence. Since it is clear that certainN-terminal shortened forms of the polymerase are active, the geneconstructs used for expression of these polymerases may also include thecorresponding shortened forms of the coding sequence.

CONTROL SEQUENCES AND CORRESPONDING HOSTS

Procaryotes most frequently are represented by various strains of E.coli. However, other microbial strains may also be used, such asbacilli, for example, Bacillus subtilis, various species of Pseudomonas,or other bacterial strains. In such procaryotic systems, plasmid vectorsthat contain replication sites and control sequences derived from aspecies compatible with the host are used. For example, E. coli istypically transformed using derivatives of pBR322, a plasmid derivedfrom an E. coli species by Bolivar, et al., Gene (1977) 2: 95. pBR322contains genes for amplicillin and tetracycline resistance, and thusprovides additional markers that can be either retained or destroyed inconstructing the desired vector. Commonly used procaryotic controlsequences, which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe β-lactamase (penicillinase) and lactose (lac) promoter systems(Chang, et al., Nature (1977) 198: 1056), the tryptophan (trp) promotersystem (Goeddel, et al., Nucleic Acids Res. (1980) 8: 4057) and thelambda-derived P_(L) promoter (Shimatake, et al., Nature (1981) 292:128) and N-gene ribosome binding site, which has been made useful as aportable control cassette (as set forth in U.S. Pat. No. 4,711,845,issued Dec. 8, 1987), which comprises a first DNA sequence that is theP_(L) promoter operably linked to a second DNA sequence corresponding toN_(RBS) upstream of a third DNA sequence having at least one restrictionsite that permits cleavage within six bp 3' of the N_(RBS) sequence.Also useful is the phosphatase A (phoA) system described by Chang, etal. in European Patent Publication No. 196,864 published Oct. 8, 1986,assigned to the same assignee and incorporated herein by reference.However, any available promoter system compatible with procaryotes canbe used.

In addition to bacteria, eucaryotic microbes, such as yeast, may also beused as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker'syeast, are most used, although a number of other strains are commonlyavailable. While vectors employing the 2 micron origin of replicationare illustrated (Broach, J. R., Meth. Enz. (1983) 101: 307), otherplasmid vectors suitable for yeast expression are known (see, forexample, Stinchcomb, et al., Nature (1979) 282: 39, Tschempe, et al.,Gene (1980) 10: 157 and Clarke, L., et al., Meth. Enz. (1983) 101: 300).Control sequences for yeast vectors include promoters for the synthesisof glycolytic enzymes (Hess, et al., J. Adv. Enzyme Reg. (1968) 7: 149;Holland, et al., Biotechnology (1978) 17: 4900).

Additional promoters known in the art include the promoter for3-phosphoglycerate kinase (Hitzeman, et al., J. Biol. Chem. (1980) 255:2073), and those for other glycolytic enzymes, such asglyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other promoters that have theadditional advantage of transcription controlled by growth conditionsare the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, and enzymes responsible for maltose and galactoseultilization (Holland, supra).

It is also believed that terminator sequences are desirable at the 3'end of the coding sequences. Such terminators are found in the 3'untranslated region following the coding sequences in yeast-derivedgenes. Many of the vectors illustrated contain control sequences derivedfrom the enolase gene containing plasmid peno46 (Holland, M. J., et al.,J. Biol. Chem. (1981) 256: 1385) or the LEU2 gene obtained from YEp13(Broach, J., et al., Gene (1978) 8: 121); however, any vector containinga yeast-compatible promoter, origin of replication, and other controlsequences is suitable.

It is also, of course, possible to express genes encoding polypeptidesin eucaryotic host cell cultures derived from multicellular organisms.See, for example, Tissue Culture, Academic Press, Cruz and Patterson,editors (1973). Useful host cell lines include murine myelomas N51, VEROand HeLa cells, and Chinese hamster ovary (CHO) cells. Expressionvectors for such cells ordinarily include promoters and controlsequences compatible with mammalian cells such as, for example, thecommonly used early and late promoters from Simian Virus 40 (SV 40)(Fiers, et al., Nature (1978) 273: 113), or other viral promoters suchas those derived from polyoma, Adenovirus 2, bovine papiloma virus, oravian sarcoma viruses, or immunoglobulin promoters and heat shockpromoters. A system for expressing DNA in mammalian systems using theBPV as a vector is disclosed in U.S. Pat. No. 4,419,446. A modificationof this system is described in U.S. Pat. No. 4,601,978. General aspectsof mammalian cell host system transformations have been described byAxel, U.S. Pat. No. 4,399,216. It now appears, also, that "enhancer"regions are important in optimizing expression; these are, generally,sequences found upstream of the promoter region. Origins of replicationmay be obtained, if needed, from viral sources. However, integrationinto the chromosome is a common mechanism for DNA replication ineucaryotes.

Plant cells are also now available as hosts, and control sequencescompatible with plant cells such as the nopaline synthase promoter andpolyadenylation signal sequences (Depicker, A., et al., J. Mol. Appl.Gen. (1982) 1: 561) are available.

Recently, in addition, expression systems employing insect cellsutilizing the control systems provided by baculovirus vectors have beendescribed (Miller, D. W., et al., in Genetic Engineering (1986) Setlow,J. K. et al., eds., Plenum Publishing, Vol. 8, pp. 277-297). Thesesystems are also successful in producing Taq polymerase.

TRANSFORMATIONS

Depending on the host cell used, transformation is done using standardtechniques appropriate to such cells. The calcium treatment employingcalcium chloride, as described by Cohen, S. N., Proc. Natl. Acad. Sci.(U.S.A.) (1972) 69: 2110 is used for procaryotes or other cells thatcontain substantial cell wall barriers. Infection with Agrobacteriumtumefaciens (Shaw, C. H., et al., Gene (1983) 23: 315) is used forcertain plant cells. For mammalian cells without such cell walls, thecalcium phosphate precipitation method of Graham and van der Eb,Virology (1978) 52: 546 is preferred. Transformations into yeast arecarried out according to the method of Van Solingen, P., et al., J.Bact. (1977) 130: 946 and Hsiao, C. L., et al., Proc. Natl. Acad. Sci.(U.S.A.) (1979) 76: 3829.

CONSTRUCTION OF A λGT11 EXPRESSION LIBRARY

The strategy for isolating DNA encoding desired proteins, such as theTaq polymerase encoding DNA, using the bacteriophage vector lambda gt11,is as follows. A library can be constructed of EcoRI-flanked AluIfragments, generated by complete digestion of Thermus aquaticus DNA,inserted at the EcoRI site in the lambda gt11 phage (Young and Davis,Proc. Natl. Acad. Sci U.S.A. (1983) 80: 1194-1198). Because the uniqueEcoRI site in this bacteriophage is located in the carboxy-terminus ofthe β-galactosidase gene, inserted DNA (in the appropriate frame andorientation) is expressed as protein fused with β-galactosidase underthe control of the lactose operon promoter/operator.

Genomic expression libraries are then screened using the antibody plaquehybridization procedure. A modification of this procedure, referred toas "epitope selection," uses antiserum against the fusion proteinsequence encoded by the phage, to confirm the identification ofhybridized plaques. Thus, this library of recombinant phages could bescreened with antibodies that recognize the 86,000-95,000 dalton Taqpolymerase in order to identify phage that carry DNA segments encodingthe antigenic determinants of this protein.

Approximately 2×10⁵ recombinant phage are screened using total rabbitTaq polymerase antiserum. In this primary screen, positive signals aredetected and one or more of these phages are purified from candidateplaques which failed to react with preimmune serum and reacted withimmune serum and analyzed in some detail. To examine the fusion proteinsproduced by the recombinant phage, lysogens of the phage in the hostY1089 are produced. Upon induction of the lysogens and gelelectrophoresis of the resulting proteins, each lysogen may be observedto produce a new protein, not found in the other lysogens, or duplicatesequences may result. Phage containing positive signals are picked; inthis case, one positive plaque was picked for further identification andreplated at lower densities to purify recombinants and the purifiedclones were analyzed by size class via digestion with EcoRI restrictionenzyme. Probes can then be made of the isolated DNA insert sequences andlabeled appropriately and these probes can be used in conventionalcolony or plaque hybridization assays described in Maniatis et al.,Molecular Cloning: A Laboratory Manual (1982), the disclosure of whichis incorporated herein by reference.

The labeled probe was used to probe a second genomic library constructedin a Charon 35 bacteriophage (Wilhelmine, A. M. et al., Gene (1983) 26:171-179). This library was made from Sau3A partial digestions of genomicThermus aquaticus DNA and size fractionated fragments (15-20 kb) werecloned into the BamHI site of the Charon 35 phage. The probe was used toisolate phage containing DNA encoding the Taq polymerase. One of theresulting phage, designated CH35:Taq#4-2, was found to contain theentire gene sequence. Partial sequences encoding portions of the genewere also isolated.

VECTOR CONSTRUCTION

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation and restriction techniquesthat are well understood in the art. Isolated plasmids, DNA sequences,or synthesized oligonucleotides are cleaved, tailored, and religated inthe form desired.

Site-specific DNA cleavage is performed by treating with the suitablerestriction enzyme (or enzymes) under conditions that are generallyunderstood in the art, and the particulars of which are specified by themanufacturer of these commercially available restriction enzymes. See,e.g., New England Biolabs, Product Catalog. In general, about 1 μg ofplasmid or DNA sequence is cleaved by one unit of enzyme in about 20 μlof buffer solution; in the examples herein, typically an excess ofrestriction enzyme is used to ensure complete digestion of the DNAsubstrate. Incubation times of about one hour to two hours at about 37°C. are workable, although variations can be tolerated. After eachincubation, protein is removed by extraction with phenol/chloroform, andmay be followed by ether extraction, and the nucleic acid recovered fromaqueous fractions by precipitation with ethanol. If desired, sizeseparation of the cleaved fragments may be performed by polyacrylamidegel or agarose gel electrophoresis using standard techniques. A generaldescription of size separations is found in Methods in Enzymology (1980)65: 499-560.

Restriction-cleaved fragments may be blunt-ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using incubation times ofabout 15 to 25 minutes at 20° to 25° C. in 50 mM Tris pH 7.6, 50 mMNaCl, 10 mM MgCl₂, 10 mM DTT and 50-100 μM dNTPs. The Klenow fragmentfills in at 5' sticky ends, but chews back protruding 3' single strands,even though the four dNTPs are present. If desired, selective repair canbe performed by supplying only one of the, or selected, dNTPs within thelimitations dictated by the nature of the sticky ends. After treatmentwith Klenow, the mixture is extracted with phenol/chloroform and ethanolprecipitated. Treatment under appropriate conditions with S1 nucleaseresults in hydrolysis of any single-stranded portion.

Synthetic oligonucleotides may be prepared using the triester method ofMatteucci, et al., (J. Am. Chem. Soc. (1981) 103: 3185-3191) or usingautomated synthesis methods. Kinasing of single strands prior toannealing or for labeling is achieved using an excess, e.g.,approximately 10 units of polynucleotide kinase to 1 nM substrate in thepresence of 50 mM Tris, pH 7.6, 10 mM MgCl₂, 5 mM dithiothreitol, 1-2 mMATP. If kinasing is for labeling of probe, the ATP will contain highspecific activity γ-³².sbsp.p.

Ligations are performed in 15-30 μl volumes under the following standardconditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 10 mMDTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either 40 μM ATP, 0.01-0.02(Weiss) units T4 DNA ligase at 0° C. (for "sticky end" ligation) or 1 mMATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C. (for "blunt end"ligation). Intermolecular "sticky end" ligations are usually performedat 33-100 μg/ml total DNA concentrations (5-100 nM total endconcentration). Intermolecular blunt end ligations (usually employing a10-30 fold molar excess of linkers) are performed at 1 μM total endsconcentration.

In vector construction employing "vector fragments", the vector fragmentis commonly treated with bacterial alkaline phosphatase (BAP) in orderto remove the 5' phosphate and prevent religation of the vector. BAPdigestions are conducted at pH 8 in approximately 150 mM Tris, in thepresence of Na⁺ and Mg⁺² using about 1 unit of BAP per mg of vector at60° C. for about one hour. In order to recover the nucleic acidfragments, the preparation is extracted with phenol/chloroform andethanol precipitated. Alternatively, religation can be prevented invectors that have been double digested by additional restriction enzymedigestion of the unwanted fragments.

MODIFICATION OF DNA SEQUENCES

For portions of vectors derived from cDNA or genomic DNA that requiresequence modifications, site-specific primer-directed mutagenesis isused. This technique is now standard in the art, and is conducted usinga synthetic oligonucleotide primer complementary to a single-strandedphage DNA to be mutagenized except for limited mismatching, representingthe desired mutation. Briefly, the synthetic oligonucleotide is used asa primer to direct synthesis of a strand complementary to the phage, andthe resulting double-stranded DNA is transformed into a phage-supportinghost bacterium. Cultures of the transformed bacteria are plated in topagar, permitting plaque formation from single cells that harbor thephage.

Theoretically, 50% of the new plaques will contain the phage having, asa single strand, the mutated form; 50% will have the original sequence.The plaques are transferred to nitrocellulose filters and the "lifts"hybridized with kinased synthetic primer at a temperature that permitshybridization of an exact match, but at which the mismatches with theoriginal strand are sufficient to prevent hybridization. Plaques thathybridize with the probe are then picked and cultured, and the DNA isrecovered.

VERIFICATION OF CONSTRUCTION

In the constructions set forth below, correct ligations for plasmidconstruction are confirmed by first transforming E. coli strain MM294,or other suitable host, with the ligation mixture. Successfultransformants are selected by ampicillin, tetracycline or otherantibiotic resistance or using other markers, depending on the mode ofplasmid construction, as is understood in the art. Plasmids from thetransformants are then prepared according to the method of Clewell, D.B., et al., Proc. Natl. Acad. Sci. (U.S.A.) (1969) 62: 1159, optionallyfollowing chloramphenicol amplification (Clewell, D. B., J. Bacteriol.(1972) 110: 667). The isolated DNA is analyzed by restriction and/orsequenced by the dideoxy method of Sanger, F., et al., Proc. Natl. Acad.Sci. (U.S.A.) (1977) 74: 5463 as further described by Messing, et al.,Nucleic Acids Res. (1981) 9: 309, or by the method of Maxam, et al.,Methods in Enzymology (1980) 65: 499.

HOST STRAINS EXEMPLIFIED

Host strains used in cloning and expression herein are as follows:

For cloning and sequencing, and for expression of constructions undercontrol of most bacterial promoters, E. coli strain MM294 obtained fromE. coli Genetic Stock Center GCSC #6135, was used as the host. Forexpression under control of the P_(L) N_(RBS) promoter, E. coli strainK12 MC1000 lambda lysogen, N₇ N₅₃ cI857 SusP₈₀, ATCC 39531 may be used.Used herein are E. coli DG116, which was deposited with ATCC (ATCC53606) on Apr. 7, 1987 and E. coli KB2, which was deposited with ATCC(ATCC 53075) on Mar. 29, 1985.

For M13 phage recombinants, E. coli strains susceptible to phageinfection, such as E. coli K12 strain DG98, are employed. The DG98strain has been deposited with ATCC July 13, 1984 and has accessionnumber 39768.

Mammalian expression can be accomplished in COS-7 COS-A2, CV-1, andmurine cells, and insect cell-based expression in Spodopterafrugipeida).

PURIFICATION

In addition to the purification procedures previously described, thethermostable polymerase of the invention may be purified usinghydrophobic interaction chromatography. Hydrophobic interactionchromatography is a separation technique in which substances areseparated on the basis of differing strengths of hydrophobic interactionwith an uncharged bed material containing hydrophobic groups. Typically,the column is first equilibrated under conditions favorable tohydrophobic binding, e.g., high ionic strength. A descending saltgradient may be used to elute the sample.

According to the invention, the aqueous mixture (containing eithernative or recombinant polymerase) is loaded onto a column containing arelatively strong hydrophobic gel such as Phenyl Sepharose (manufacturedby Pharmacia) or Phenyl TSK (manufactured by Toyo Soda). To promotehydrophobic interaction with a Phenyl Sepharose column, a solvent isused which contains, for example, greater than or equal to 0.2M ammoniumsulfate, with 0.2M being preferred. Thus the column and the sample areadjusted to 0.2M ammonium sulfate in 50 mM Tris-1 mM EDTA buffer and thesample applied to the column. The column is washed with the 0.2Mammonium sulfate buffer. The enzyme may then be eluted with solventswhich attenuate hydrophobic interactions such as, for example,decreasing salt gradients, ethylene or propylene glycol, or urea. Forthe recombinant Taq polymerase, a preferred embodiment involves washingthe column sequentially with the Tris-EDTA buffer and the Tris-EDTAbuffer containing 20% ethylene glycol. The Taq polymerase issubsequently eluted from the column with a 0-4M urea gradient in theTris-EDTA ethylene glycol buffer.

STABILIZATION OF ENZYME ACTIVITY

For long-term stability, the enzyme herein must be stored in a bufferthat contains one or more non-ionic polymeric detergents. Suchdetergents are generally those that have a molecular weight in the rangeof approximately 100 to 250,000, preferably about 4,000 to 200,000daltons and stabilize the enzyme at a pH of from about 3.5 to about 9.5,preferably from about 4 to 8.5. Examples of such detergents includethose specified on pages 295-298 of McCutcheon's Emulsifiers &Detergents, North American edition (1983), published by the McCutcheonDivision of MC Publishing Co., 175 Rock Road, Glen Rock, N.J. (U.S.A.),the entire disclosure of which is incorporated herein by reference.Preferably, the detergents are selected from the group comprisingethoxylated fatty alcohol ethers and lauryl ethers, ethoxylated alkylphenols, octylphenoxy polyethoxy ethanol compounds, modifiedoxyethylated and/or oxypropylated straight-chain alcohols, polyethyleneglycol monooleate compounds, polysorbate compounds, and phenolic fattyalcohol ethers. More particularly preferred are Tween 20, from ICIAmericas Inc., Wilmington, Del., which is a polyoxyethylated (20)sorbitan monolaurate, and Iconol™ NP-40, from BASF Wyandotte Corp.Parsippany, N.J., which is an ethoxylated alkyl phenol (nonyl).

The thermostable enzyme of this invention may be used for any purpose inwhich such enzyme is necessary or desirable. In a particularly preferredembodiment, the enzyme herein is employed in the amplification protocolset forth below.

AMPLIFICATION PROTOCOL

The amplification protocol using the enzyme of the present invention maybe the process for amplifying existing nucleic acid sequences that isdisclosed and claimed in U.S. Pat. No. 4,683,202, issued July 28, 1987,the disclosure of which is incorporated herein by reference. Preferably,however, the enzyme herein is used in the amplification processdisclosed below.

Specifically, the amplification method involves amplifying at least onespecific nucleic acid sequence contained in a nucleic acid or a mixtureof nucleic acids, wherein if the nucleic acid is double-stranded, itconsists of two separated complementary strands of equal or unequallength, which process comprises:

(a) contacting each nucleic acid strand with four different nucleotidetriphosphates and one oligonucleotide primer for each different specificsequence being amplified, wherein each primer is selected to besubstantially complementary to different strands of each specificsequence, such that the extension product synthesized from one primer,when it is separated from its complement, can serve as a template forsynthesis of the extension product of the other primer, said contactingbeing at a temperature which promotes hybridization of each primer toits complementary nucleic acid strand;

(b) contacting each nucleic acid strand, at the same time as or afterstep (a), with a DNA polymerase from Thermus aquaticus which enablescombination of the nucleotide triphosphates to form primer extensionproducts complementary to each strand of each nucleic acid;

(c) maintaining the mixture from step (b) at an effective temperaturefor an effective time to promote the activity of the enzyme, and tosynthesize, for each different sequence being amplified, an extensionproduct of each primer which is complementary to each nucleic acidstrand template, but not so high as to separate each extension productfrom its complementary strand template;

(d) heating the mixture from step (c) for an effective time and at aneffective temperature to separate the primer extension products from thetemplates on which they were synthesized to produce single-strandedmolecules, but not so high as to denature irreversibly the enzyme;

(e) cooling the mixture from step (d) for an effective time and to aneffective temperature to promote hybridization of each primer to each ofthe single-stranded molecules produced in step (d); and

(f) maintaining the mixture from step (e) at an effective temperaturefor an effective time to promote the activity of the enzyme and tosynthesize, for each different sequence being amplified, an extensionproduct of each primer which is complementary to each nucleic acidstrand template produced in step (d), but not so high as to separateeach extension product from its complementary strand template whereinthe effective time and temperatures in steps (e) and (f) may coincide(steps (e) and (f) are carried out simultaneously), or may be separate.

Steps (d)-(f) may be repeated until the desired level of sequenceamplification is obtained.

The amplification method is useful not only for producing large amountsof an existing completely specified nucleic acid sequence, but also forproducing nucleic acid sequences which are known to exist but are notcompletely specified. In either case an initial copy of the sequence tobe amplified must be available, although it need not be pure or adiscrete molecule.

In general, the amplification process involves a chain reaction forproducing, in exponential quantities relative to the number of reactionsteps involved, at least one specific nucleic acid sequence given (a)that the ends of the required sequence are known in sufficient detailthat oligonucleotides can be synthesized which will hybridize to them,and (b) that a small amount of the sequence is available to initiate thechain reaction. The product of the chain reaction will be a discretenucleic acid duplex with termini corresponding to the ends of thespecific primers employed.

Any nucleic acid sequence, in purified or nonpurified form, can beutilized as the starting nucleic acid(s), provided it contains or issuspected to contain the specific nucleic acid sequence desired. Thus,the process may employ, for example, DNA or RNA, including messengerRNA, which DNA or RNA may be single-stranded or double-stranded. Inaddition, a DNA-RNA hybrid which contains one strand of each may beutilized. A mixture of any of these nucleic acids may also be employed,or the nucleic acids produced from a previous amplification reactionherein using the same or different primers may be so utilized. Thespecific nucleic acid sequence to be amplified may be only a fraction ofa larger molecule or can be present initially as a discrete molecule, sothat the specific sequence constitutes the entire nucleic acid.

It is not necessary that the sequence to be amplified be presentinitially in a pure form; it may be a minor fraction of a complexmixture, such as a portion of the β-globin gene contained in whole humanDNA (as exemplified in Saiki et al., Science, 230, 1530-1534 (1985)) ora portion of a nucleic acid sequence due to a particular microorganismwhich organism might constitute only a very minor fraction of aparticular biological sample. The starting nucleic acid sequence maycontain more than one desired specific nucleic acid sequence which maybe the same or different. Therefore, the amplification process is usefulnot only for producing large amounts of one specific nucleic acidsequence, but also for amplifying simultaneously more than one differentspecific nucleic acid sequence located on the same or different nucleicacid molecules.

The nucleic acid(s) may be obtained from any source, for example, fromplasmids such as pBR322, from cloned DNA or RNA, or from natural DNA orRNA from any source, including bacteria, yeast, viruses, organelles, andhigher organisms such as plants or animals. DNA or RNA may be extractedfrom blood, tissue material such as chorionic villi, or amniotic cellsby a variety of techniques such as that described by Maniatis et al.,supra, p. 280-281.

If probes are used which are specific to a sequence being amplified andthereafter detected, the cells may be directly used without extractionof the nucleic acid if they are suspended in hypotonic buffer and heatedto about 90°-100° C., until cell lysis and dispersion of intracellularcomponents occur, generally 1 to 15 minutes. After the heating step theamplification reagents may be added directly to the lysed cells.

Any specific nucleic acid sequence can be produced by the amplificationprocess. It is only necessary that a sufficient number of bases at bothends of the sequence be known in sufficient detail so that twooligonucleotide primers can be prepared which will hybridize todifferent strands of the desired sequence and at relative positionsalong the sequence such that an extension product synthesized from oneprimer, when it is separated from its template (complement), can serveas a template for extension of the other primer into a nucleic acidsequence of defined length. The greater the knowledge about the bases atboth ends of the sequence, the greater can be the specificity of theprimers for the target nucleic acid sequence, and thus the greater theefficiency of the process.

It will be understood that the word "primer" as used hereinafter mayrefer to more than one primer, particularly in the case where there issome ambiguity in the information regarding the terminal sequence(s) ofthe fragment to be amplified. For instance, in the case where a nucleicacid sequence is inferred from protein sequence information, acollection of primers containing sequences representing all possiblecodon variations based on degeneracy of the genetic code will be usedfor each strand. One primer from this collection will be homologous withthe end of the desired sequence to be amplified.

The oligonucleotide primers may be prepared using any suitable method,such as, for example, the phosphotriester and phosphodiester methodsdescribed above, or automated embodiments thereof. In one such automatedembodiment, diethylphosphoramidites are used as starting materials andmay be synthesized as described by Beaucage et al., Tetrahedron Letters(1981), 22: 1859-1862. One method for synthesizing oligonucleotides on amodified solid support is described in U.S. Pat. No. 4,458,066. It isalso possible to use a primer which has been isolated from a biologicalsource (such as a restriction endonuclease digest).

The specific nucleic acid sequence is produced by using the nucleic acidcontaining that sequence as a template. The first step involvescontacting each nucleic acid strand with four different nucleotidetriphosphates and one oligonucleotide primer for each different nucleicacid sequence being amplified or detected. If the nucleic acids to beamplified or detected are DNA, then the nucleotide triphosphates aredATP, dCTP, dGTP and TTP.

The nucleic acid strands are used as a template for the synthesis ofadditional nucleic acid strands. This synthesis can be performed usingany suitable method. Generally it occurs in a buffered aqueous solution,preferably at a pH of 7-9, most preferably about 8. Preferably, a molarexcess (for cloned nucleic acid, usually about 1000:1 primer:template,and for genomic nucleic acid, usually about 10⁸ :1 primer:template) ofthe two oligonucleotide primers is added to the buffer containing theseparated template strands. It is understood, however, that the amountof complementary strand may not be known if the process herein is usedfor diagnostic applications, so that the amount of primer relative tothe amount of complementary strand cannot be determined with certainty.As a practical matter, however, the amount of primer added willgenerally be in molar excess over the amount of complementary strand(template) when the sequence to be amplified is contained in a mixtureof complicated long-chain nucleic acid strands. A large molar excess ispreferred to improve the efficiency of the process.

Preferably the concentration of nucleotide triphosphates is 150-200 μMeach in the buffer for amplification and MgCl₂ is present in the bufferin an amount of 1.5-2 mM to increase the efficiency and specificity ofthe reaction.

The resulting solution is then treated according to whether the nucleicacids being amplified or detected are double or single-stranded. If thenucleic acids are single-stranded, then no denaturation step need beemployed, and the reaction mixture is held at a temperature whichpromotes hybridization of the primer to its complementary target(template) sequence. Such temperature is generally from about 35° C. to65° C. or more, preferably about 37°-60° C. for an effective time,generally one-half to five minutes, preferably one-three minutes.Preferably, 45°-58° C. is used for Taq polymerase and >15-mer primers toincrease the specificity of primer hybridization. Shorter primers needlower temperatures.

The complement to the original single-stranded nucleic acid may besynthesized by adding one or two oligonucleotide primers thereto. If anappropriate single primer is added, a primer extension product issynthesized in the presence of the primer, the DNA polymerase fromThermus aquaticus and the nucleotide triphosphates. The product will bepartially complementary to the single-stranded nucleic acid and willhybridize with the nucleic acid strand to form a duplex of strands ofunequal length which may then be separated into single strands asdescribed above to produce two single separated complementary strands.Alternatively, two appropriate primers may be added to thesingle-stranded nucleic acid and the reaction carried out.

If the nucleic acid contains two strands, it is necessary to separatethe strands of the nucleic acid before it can be used as the template.This strand separation can be accomplished by any suitable denaturingmethod including physical, chemical or enzymatic means. One preferredphysical method of separating the strands of the nucleic acid involvesheating the nucleic acid until it is completely (>99%) denatured.Typical heat denaturation involves temperatures ranging from about 90°to 105° C. for times generally ranging from about 0.5 to 5 minutes.Preferably the effective denaturing temperature is 90°-100° C. for 0.5to 3 minutes. Strand separation may also be induced by an enzyme fromthe class of enzymes known as helicases or the enzyme RecA, which hashelicase activity and in the presence of riboATP is known to denatureDNA. The reaction conditions suitable for separating the strands ofnucleic acids with helicases are described by Kuhn Hoffmann-Berling,CSH-Quantitative Biology, 43: 63 (1978), and techniques for using RecAare reviewed in C. Radding, Ann. Rev. Genetics, 16: 405-37 (1982). Thedenaturation produces two separated complementary strands of equal orunequal length.

If the double-stranded nucleic acid is denatured by heat, the reactionmixture is allowed to cool to a temperature which promotes hybridizationof each primer present to its complementary target (template) sequence.This temperature is usually from about 35° C. to 65° C. or more,depending on reagents, preferably 37°-60° C., maintained for aneffective time, generally 0.5 to 5 minutes, and preferably 1-3 minutes.In practical terms, the temperature is simply lowered from about 95° C.to as low as 37° C., preferably to about 45°-58° C. for Taq polymerase,and hybridization occurs at a temperature within this range.

Whether the nucleic acid is single- or double-stranded, the DNApolymerase from Thermus aquaticus may be added at the denaturation stepor when the temperature is being reduced to or is in the range forpromoting hybridization. The reaction mixture is then heated to atemperature at which the activity of the enzyme is promoted oroptimized, i.e., a temperature sufficient to increase the activity ofthe enzyme in facilitating synthesis of the primer extension productsfrom the hybridized primer and template. The temperature must actuallybe sufficient to synthesize an extension product of each primer which iscomplementary to each nucleic acid template, but must not be so high asto denature each extension product from its complementary template(i.e., the temperature is generally less than about 80° C.-90° C.).

Depending mainly on the types of enzyme and nucleic acid(s) employed,the typical temperature effective for this synthesis reaction generallyranges from about 40° to 80° C., preferably 50°-75° C. The temperaturemore preferably ranges from about 65°-75° C. when a DNA polymerase fromThermus aquaticus is employed. The period of time required for thissynthesis may range from about 0.5 to 40 minutes or more, dependingmainly on the temperature, the length of the nucleic acid, the enzymeand the complexity of the nucleic acid mixture, preferably one to threeminutes. If the nucleic acid is longer, a longer time period isgenerally required. The presence of dimethylsulfoxide (DMSO) is notnecessary or recommended because DMSO was found to inhibit Taqpolymerase enzyme activity.

The newly synthesized strand and its complementary nucleic acid strandform a double-stranded molecule which is used in the succeeding steps ofthe process. In the next step, the strands of the double-strandedmolecule are separated by heat denaturation at a temperature effectiveto denature the molecule, but not so high that the thermostable enzymeis completely and irreversibly denatured or inactivated. Dependingmainly on the type of enzyme and the length of nucleic acid, thistemperature generally ranges from about 90° to 105° C., more preferably90°-100° C., and the time for denaturation typically ranges from 0.5 tofour minutes, depending mainly on the temperature and nucleic acidlength.

After this time, the temperature is decreased to a level which promoteshybridization of the primer to its complementary single-strandedmolecule (template) produced from the previous step. Such temperature isdescribed above.

After this hybridization step, or in lieu of (or concurrently with) thehybridization step, the temperature is adjusted to a temperature that iseffective to promote the activity of the thermostable enzyme to enablesynthesis of a primer extension product using as template the newlysynthesized strand from the previous step. The temperature again mustnot be so high as to separate (denature) the extension product from itstemplate, as previously described (usually from 40° to 80° C. for 0.5 to40 minutes, preferably 50° to 70° C. for one-three minutes).Hybridization may occur during this step, so that the previous step ofcooling after denaturation is not required. In such a case, usingsimultaneous steps, the preferred temperature range is 50°-70° C.

The heating and cooling steps of strand separation, hybridization, andextension product synthesis can be repeated as often as needed toproduce the desired quantity of the specific nucleic acid sequence,depending on the ultimate use. The only limitation is the amount of theprimers, thermostable enzyme and nucleotide triphosphates present.Preferably, the steps are repeated at least twice. For use in detection,the number of cycles will depend, e.g., on the nature of the sample. Forexample, fewer cycles will be required if the sample being amplified ispure. If the sample is a complex mixture of nucleic acids, more cycleswill be required to amplify the signal sufficiently for its detection.For general amplification and detection, preferably the process isrepeated at least 20 times.

When labeled sequence-specific probes are employed as described below,preferably the steps are repeated at least five times. When humangenomic DNA is employed with such probes, the process is repeatedpreferably 15-30 times to amplify the sequence sufficiently that aclearly detectable signal is produced, i.e., so that background noisedoes not interfere with detection.

As will be described in further detail below, the amount of the specificnucleic acid sequence produced will accumulate in an exponentialfashion.

No additional nucleotides, primers, or thermostable enzyme need be addedafter the initial addition, provided that the enzyme has not becomedenatured or inactivated irreversibly, in which case it is necessary toreplenish the enzyme after each denaturing step. Addition of suchmaterials at each step, however, will not adversely affect the reaction.

When it is desired to produce more than one specific nucleic acidsequence from the first nucleic acid or mixture of nucleic acids, theappropriate number of different oligonucleotide primers are utilized.For example, if two different specific nucleic acid sequences are to beproduced, four primers are utilized. Two of the primers are specific forone of the specific nucleic acid sequences and the other two primers arespecific for the second specific nucleic acid sequence. In this manner,each of the two different specific sequences can be producedexponentially by the present process.

After the appropriate length of time has passed to produce the desiredamount of the specific nucleic acid sequence, the reaction may be haltedby inactivating the enzyme in any known manner (e.g., by adding EDTA,phenol, SDS or CHCl₃) or by separating the components of the reaction.

The amplification process may be conducted continuously. In oneembodiment of an automated process, the reaction mixture may betemperature cycled such that the temperature is programmed to becontrolled at a certain level for a certain time.

One such instrument for this purpose is the automated machine forhandling the amplification reaction of this invention described in nowabandoned Ser. No. 833,368 filed Feb. 25, 1986 entitled "Apparatus AndMethod For Performing Automated Amplification of Nucleic Acid SequencesAnd Assays Using Heating And Cooling Steps," the disclosure of which isincorporated herein by reference. Briefly, this instrument utilizes aliquid handling system under computer control to make liquid transfersof enzyme stored at a controlled temperature in a first receptacle intoa second receptacle whose temperature is controlled by the computer toconform to a certain incubation profile. The second receptacle storesthe nucleic acid sequence(s) to be amplified plus the nucleotidetriphosphates and primers. The computer includes a user interfacethrough which a user can enter process parameters that control thecharacteristics of the various steps in the amplification sequence suchas the times and temperatures of incubation, the amount of enzyme totransfer, etc.

A preferred machine that may be employed utilizes temperature cyclingwithout a liquid handling system because the enzyme need not betransferred at every cycle. Such a machine is described more completelyin European Patent Application No. 236,069, published Sept. 9, 1987, thedisclosure of which is incorporated herein by reference. Briefly, thisinstrument consists of the following systems:

1. A heat-conducting container for holding a given number of tubes,preferably 500 μl tubes, which contain the reaction mixture ofnucleotide triphosphates, primers, nucleic acid sequences, and enzyme.

2. A means to heat, cool, and maintain the heat-conducting containerabove and below room temperature, which means has an input for receivinga control signal for controlling which of the temperatures at or towhich the container is heated, cooled or maintained. (These may bePeltier heat pumps available from Materials Electronics ProductsCorporation in Trenton, N.J. or a water heat exchanger.)

3. A computer means (e.g., a microprocessor controller), coupled to theinput of said means, to generate the signals that control automaticallythe amplification sequence, the temperature levels, and the temperatureramping and timing.

The amplification protocol is demonstrated diagrammatically below, wheredouble-stranded DNA containing the desired sequence [S] comprised ofcomplementary strands [S⁺ ] and [S⁻ ] is utilized as the nucleic acid.During the first and each subsequent reaction cycle, extension of eacholigonucleotide primer on the original template will produce one newssDNA molecule product of indefinite length that terminates with onlyone of the primers. These products, hereafter referred to as "longproducts," will accumulate in a linear fashion; that is, the amountpresent after any number of cycles will be proportional to the number ofcycles.

The long products thus produced will act as templates for one or theother of the oligonucleotide primers during subsequent cycles and willproduce molecules of the desired sequence [S⁺ ] or [S⁻ ]. Thesemolecules will also function as templates for one or the other of theoligonucleotide primers, producing further [S⁺ ] and [S⁻ ], and thus achain reaction can be sustained that will result in the accumulation of[S] at an exponential rate relative to the number of cycles.

By-products formed by oligonucleotide hybridizations other than thoseintended are not self-catalytic (except in rare instances) and thusaccumulate at a linear rate.

The specific sequence to be amplified, [S], can be depicteddiagrammatically as: ##STR1## The appropriate oligonucleotide primerswould be: ##STR2## so that if DNA containing [S]

    . . . zzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz . . .

    . . . zzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGzzzzzzzzzzzzzzzzz . . .

is separated into single strands and its single strands are hybridizedto Primers 1 and 2, the following extension reactions can be catalyzedby a thermostable polymerase in the presence of the four nucleotidetriphosphates: ##STR3##

On denaturation of the two duplexes formed, the products are: ##STR4##If these four strands are allowed to rehybridize with Primers 1 and 2 inthe next cycle, the thermostable polymerase will catalyze the followingreactions: ##STR5##

If the strands of the above four duplexes are separated, the followingstrands are found: ##STR6##

It is seen that each strand which terminates with the oligonucleotidesequence of one primer and the complementary sequence of the other isthe specific nucleic acid sequence [S] that is desired to be produced.

The amount of original nucleic acid remains constant in the entireprocess, because it is not replicated. The amount of the long productsincreases linearly because they are produced only from the originalnucleic acid. The amount of the specific sequence increasesexponentially. Thus, the specific sequence will become the predominantspecies. This is illustrated in the following table, which indicates therelative amounts of the species theoretically present after n cycles,assuming 100% efficiency at each cycle:

    ______________________________________                                        Number of Double Strands                                                      After 0 to n Cycles                                                                                Long     Specific                                        Cycle Number                                                                            Template   Products Sequence [S]                                    ______________________________________                                        0         1          --       --                                              1         1          1        0                                               2         1          2        1                                               3         1          3        4                                               5         1          5        26                                              10        1          10       1013                                            15        1          15       32,752                                          20        1          20       1,048,555                                       n         1          n        (2.sup.n -n-1)                                  ______________________________________                                    

When a single-stranded nucleic acid is utilized as the template, onlyone long product is formed per cycle.

A sequence within a given sequence can be amplified after a given numberof amplifications to obtain greater specificity of the reaction byadding after at least one cycle of amplification a set of primers thatare complementary to internal sequences (that are not on the ends) ofthe sequence to be amplified. Such primers may be added at any stage andwill provide a shorter amplified fragment. Alternatively, a longerfragment can be prepared by using primers with non-complementary endsbut having some overlap with the primers previously utilized in theamplification.

The amplification method may be utilized to clone a particular nucleicacid sequence for insertion into a suitable expression vector. Thevector may be used to transform an appropriate host organism to producethe gene product of the sequence by standard methods of recombinant DNAtechnology. Such cloning may involve direct ligation into a vector usingblunt-end ligation, or use of restriction enzymes to cleave at sitescontained within the primers.

In addition, the amplification process can be used for in vitromutagenesis. The oligodeoxyribonucleotide primers need not be exactlycomplementary to the DNA sequence that is being amplified. It is onlynecessary that they be able to hybridize to the sequence sufficientlywell to be extended by the thermostable enzyme. The product of anamplification reaction wherein the primers employed are not exactlycomplementary to the original template will contain the sequence of theprimer rather than the template, thereby introducing an in vitromutation. In further cycles this mutation will be amplified with anundiminished efficiency because no further mispaired priming isrequired. The mutant thus produced may be inserted into an appropriatevector by standard molecular biological techniques and might confermutant properties on this vector such as the potential for production ofan altered protein.

The process of making an altered DNA sequence as described above couldbe repeated on the altered DNA using different primers to induce furthersequence changes. In this way, a series of mutated sequences couldgradually be produced wherein each new addition to the series coulddiffer from the last in a minor way, but from the original DNA sourcesequence in an increasingly major way. In this manner, changes could bemade ultimately which were not feasible in a single step due to theinability of a very seriously mismatched primer to function.

In addition, the primer can contain as part of its sequence anon-complementary sequence, provided that a sufficient amount of theprimer contains a sequence that is complementary to the strand to beamplified. For example, a nucleotide sequence that is not complementaryto the template sequence (such as, e.g., a promoter, linker, codingsequence, etc.) may be attached at the 5' end of one or both of theprimers, and thereby appended to the product of the amplificationprocess. After the extension primer is added, sufficient cycles are runto achieve the desired amount of new template containing thenon-complementary nucleotide insert. This allows production of largequantities of the combined fragments in a relatively short period oftime (e.g., two hours or less) using a simple technique.

The amplification method may also be used to enable detection and/orcharacterization of specific nucleic acid sequences associated withinfectious diseases, genetic disorders or cellular disorders such ascancer, e.g., oncogenes. Amplification is useful when the amount ofnucleic acid available for analysis is very small, as, for example, inthe prenatal diagnosis of sickle cell anemia using DNA obtained fromfetal cells. Amplification is particularly useful if such an analysis isto be done on a small sample using non-radioactive detection techniqueswhich may be inherently insensitive, or where radioactive techniques arebeing employed, but where rapid detection is desirable.

For the purposes of this discussion, genetic diseases may includespecific deletions and/or mutations in genomic DNA from any organism,such as, e.g., sickle cell anemia, cystic fibrosis, α-thalassemia,β-thalassemia, and the like. Sickle cell anemia can be readily detectedvia oligomer restriction analysis as described by EP Patent Publication164,054 published Dec. 11, 1985, or via a RFLP-like analysis followingamplification of the appropriate DNA sequence by the amplificationmethod. α-Thalassemia can be detected by the absence of a sequence, andβ-thalassemia can be detected by the presence of a polymorphicrestriction site closely linked to a mutation that causes the disease.

All of these genetic diseases may be detected by amplifying theappropriate sequence and analyzing it by Southern blots without usingradioactive probes. In such a process, for example, a small sample ofDNA from, e.g., amniotic fluid containing a very low level of thedesired sequence is amplified, cut with a restriction enzyme, andanalyzed via a Southern blotting technique. The use of non-radioactiveprobes is facilitated by the high level of the amplified signal.

In another embodiment, a small sample of DNA may be amplified to aconvenient level and then a further cycle of extension reactionsperformed wherein nucleotide derivatives which are readily detectable(such as ³² P-labeled or biotin-labeled nucleotide triphosphates) areincorporated directly into the final DNA product, which may be analyzedby restriction and electrophoretic separation or any other appropriatemethod.

In a further embodiment, the nucleic acid may be exposed to a particularrestriction endonuclease prior to amplification. Since a sequence whichhas been cut cannot be amplified, the appearance of an amplifiedfragment, despite prior restriction of the DNA sample, implies theabsence of a site for the endonuclease within the amplified sequence.The presence or absence of an amplified sequence can be detected by anappropriate method.

A practical application of the amplification technique, that is, infacilitating the detection of sickle cell anemia via the oligomerrestriction technique [described in EP 164,054, supra, and by Saiki etal., Bio/Technology, Vol. 3, pp. 1008-1012 (1985)] is described indetail in the Saiki et al. Science article cited above. In that Sciencearticle, a specific amplification protocol is exemplified using aβ-globin gene segment.

The amplification method herein may also be used to detect directlysingle-nucleotide variations in nucleic acid sequence (such as genomicDNA) using sequence-specific oligonucleotides, as described more fullyin European Patent Publication 237,362, published Sept. 16, 1987, thedisclosure of which is incorporated herein by reference.

Briefly, in this process, the amplified sample is spotted directly on aseries of membranes, and each membrane is hybridized with a differentlabeled sequence-specific oligonucleotide probe. After hydridization thesample is washed and the label is detected. This technique is especiallyuseful in detecting DNA polymorphisms.

Various infectious diseases can be diagnosed by the presence in clinicalsamples of specific DNA sequences characteristic of the causativemicroorganism. These include bacteria, such as Salmonella, Chlamydia,Neisseria; viruses, such as the hepatitis viruses, and parasites, suchas the Plasmodium responsible for malaria. U.S. Patent ReexaminationCertificate B1 4,358,535 issued to Falkow et al. on May 13, 1986describes the use of specific DNA hybridization probes for the diagnosisof infectious diseases. A relatively small number of pathogenicorganisms may be present in a clinical sample from an infected patientand the DNA extracted from these may constitute only a very smallfraction of the total DNA in the sample. Specific amplification ofsuspected pathogen-specific sequences prior to immobilization anddetection by hybridization of the DNA samples could greatly improve thesensitivity and specificity of traditional procedures.

Routine clinical use of DNA probes for the diagnosis of infectiousdiseases would be simplified considerably if non-radioactively labeledprobes could be employed as described in EP 63,879 to Ward. In thisprocedure biotin-containing DNA probes are detected by chromogenicenzymes linked to avidin or biotin-specific antibodies. This type ofdetection is convenient, but relatively insensitive. The combination ofspecific DNA amplification by the present method and the use of stablylabeled probes could provide the convenience and sensitivity required tomake the Falkow et al. and Ward procedures useful in a routine clinicalsetting.

A specific use of the amplification technology for detecting ormonitoring for the AIDS virus is described in European PatentPublication 229,701, published July 22, 1987, the disclosure of which isincorporated herein by reference. Briefly, the amplification anddetection process is used with primers and probes which are designed toamplify and detect, respectively, nucleic acid sequences that aresubstantially conserved among the nucleic acids in AIDS viruses andspecific to the nucleic acids in AIDS viruses. Thus, the sequence to bedetected must be sufficiently complementary to the nucleic acids in AIDSviruses to initiate polymerization preferably at room temperature in thepresence of the enzyme and nucleotide triphosphates.

A preferred amplification process described in U.S. Ser. No. 07/076,394,filed July 22, 1987, assigned to the same assignee, and incorporatedherein by reference, uses labeled primers. The label on the amplifiedproduct may be used to "capture" or immobilize the product forsubsequent detection (e.g., biotinylated amplification primers yieldlabeled products that can be "captured" by their interaction with avidinor strepavidin). As demonstrated in the aforementioned amplificationprotocols, the extension product of one labeled primer when hybridizedto the other becomes a template for the production of the desiredspecific nucleic acid sequence, and vice versa, and the process isrepeated as often as necessary to produce the desired amount of thesequence. Examples of specific preferred reagents that can be employedas the label are provided in U.S. Pat. No. 4,582,789, the disclosure ofwhich is incorporated herein by reference.

The amplification process can also be utilized to produce sufficientquantities of DNA from a single copy human gene such that detection by asimple non-specific DNA stain such as ethidium bromide can be employedto diagnose DNA directly.

In addition to detecting infectious diseases and pathologicalabnormalities in the genome of organisms, the amplification process canalso be used to detect DNA polymorphisms which may not be associatedwith any pathological state.

In summary, the amplification process is seen to provide a process foramplifying one or more specific nucleic acid sequences using a chainreaction and a thermostable enzyme, in which reaction primer extensionproducts are produced which can subsequently act as templates forfurther primer extension reactions. The process is especially useful indetecting nucleic acid sequences which are initially present in onlyvery small amounts.

The following examples are offered by way of illustration only and areby no means intended to limit the scope of the claimed invention. Inthese examples, all percentages are by weight if for solids and byvolume if for liquids, unless otherwise noted, and all temperatures aregiven in degrees Celsius.

EXAMPLE I A. Synthesis of the Primers

The following two oligonucleotide primers were prepared by the methoddescribed below:

    5'-ACACAACTGTGTTCACTAGC-3'(PC03)

    5'-CAACTTCATCCACGTTCACC-3'(PC04)

These primers, both 20-mers, anneal to opposite strands of the genomicDNA with their 5' ends separated by a distance of 110 base pairs.

1. Automated Synthesis Procedures: The diethylphosphoramidites,synthesized according to Beaucage and Caruthers (Tetrahedron Letters(1981) 22:1859-1862) were sequentially condensed to a nucleosidederivatized controlled pore glass support using a Biosearch SAM-1. Theprocedure included detritylation with trichloroacetic acid indichloromethane, condensation using benzotriazole as activating protondonor, and capping with acetic anhydride and dimethylaminopyridine intetrahydrofuran and pyridine. Cycle time was approximately 30 minutes.Yields at each step were essentially quantitative and were determined bycollection and spectroscopic examination of the dimethoxytrityl alcoholreleased during detritylation.

2. Oligodeoxyribonucleotide Deprotection and Purification Procedures:The solid support was removed from the column and exposed to 1 mlconcentrated ammonium hydroxide at room temperature for four hours in aclosed tube. The support was then removed by filtration and the solutioncontaining the partially protected oligodeoxynucleotide was brought to55° C. for five hours. Ammonia was removed and the residue was appliedto a preparative polyacrylamide gel. Electrophoresis was carried out at30 volts/cm for 90 minutes after which the band containing the productwas identified by UV shadowing of a fluorescent plate. The band wasexcised and eluted with 1 ml distilled water overnight at 4° C. Thissolution was applied to an Altech RP18 column and eluted with a 7-13%gradient of acetonitrile in 1% ammonium acetate buffer at pH 6.0. Theelution was monitored by UV absorbance at 260 nm and the appropriatefraction collected, quantitated by UV absorbance in a fixed volume andevaporated to dryness at room temperature in a vacuum centrifuge.

3. Characterization of Oligodeoxyribonucleotides: Test aliquots of thepurified oligonucleotides were ³² P labeled with polynucleotide kinaseand γ-³² P-ATP. The labeled compounds were examined by autoradiographyof 14-20% polyacrylamide gels after electrophoresis for 45 minutes at 50volts/cm. This procedure verifies the molecular weight. Base compositionwas determined by digestion of the oligodeoxyribonucleotide tonucleosides by use of venom diesterase and bacterial alkalinephosphatase and subsequent separation and quantitation of the derivednucleosides using a reverse phase HPLC column and a 10% acetonitrile, 1%ammonium acetate mobile phase.

B. Isolation of Human Genomic DNA from Cell Line

High molecular weight genomic DNA was isolated from a T cell line, Molt4, homozygous for normal β-globin available from the Human GeneticMutant Cell Depository, Camden, N.J. as GM2219C using essentially themethod of Maniatis et al., supra, p. 280-281.

C. Purification of a Polymerase From Thermus aquaticus

Thermus aquaticus strain YT1, available without restriction from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.,as ATCC No. 25,104 was grown in flasks in the following medium:

    ______________________________________                                        Sodium Citrate         1      mM                                              Potassium Phosphate, pH 7.9                                                                          5      mM                                              Ammonium Chloride      10     mM                                              Magnesium Sulfate      0.2    mM                                              Calcium Chloride       0.1    mM                                              Sodium Chloride        1      g/l                                             Yeast Extract          1      g/l                                             Tryptone               1      g/l                                             Glucose                2      g/l                                             Ferrous Sulfate        0.01   mM                                              ______________________________________                                    

(The pH was adjusted to 8.0 prior to autoclaving.)

A 10-liter fermentor was inoculated from a seed flask cultured overnightin the above medium at 70° C. A total of 600 ml from the seed flask wasused to inoculate 10 liters of the same medium. The pH was controlled at8.0 with ammonium hydroxide with the dissolved oxygen at 40%, with thetemperature at 70° C., and with the stirring rate at 400 rpm.

After growth of the cells, they were purified using the protocol (withslight modification) of Kaledin et al., supra, through the first fivestages and using a different protocol for the sixth stage. All six stepswere conducted at 4° C. The rate of fractionation on columns was 0.5columns/hour and the volumes of gradients during elution were 10 columnvolumes. An alternative and preferred purification protocol is providedin Example XIII below.

Briefly, the above culture of the T. aquaticus cells was harvested bycentrifugation after nine hours of cultivation, in late log phase, at acell density of 1.4 g dry weight/l. Twenty grams of cells wereresuspended in 80 ml of a buffer consisting of 50 mM Tris.HCl pH 7.5,0.1 mM EDTA. Cells were lysed and the lysate was centrifuged for twohours at 35,000 rpm in a Beckman TI 45 rotor at 4° C. The supernatantwas collected (fraction A) and the protein fraction precipitatingbetween 45 and 75% saturation of ammonium sulfate was collected,dissolved in a buffer consisting of 0.2M potassium phosphate buffer, pH6.5, 10 mM 2-mercaptoethanol, and 5% glycerine, and finally dialyzedagainst the same buffer to yield fraction B.

Fraction B was applied to a 2.2×30-cm column of DEAE-cellulose,equilibrated with the above described buffer. The column was then washedwith the same buffer and the fractions containing protein (determined byabsorbance at 280 nm) were collected. The combined protein fraction wasdialyzed against a second buffer, containing 0.01M potassium phosphatebuffer, pH 7.5, 10 mM 2-mercaptoethanol, and 5% glycerine, to yieldfraction C.

Fraction C was applied to a 2.6×21-cm column of hydroxyapatite,equilibrated with a second buffer. The column was then washed and theenzyme was eluted with a linear gradient of 0.01-0.5M potassiumphosphate buffer, pH 7.5, containing 10 mM 2-mercaptoethanol and 5%glycerine. Fractions containing DNA polymerase activity (90-180 mMpotassium phosphate) were combined, concentrated four-fold using anAmicon stirred cell and YM10 membrane, and dialyzed against the secondbuffer to yield fraction D.

Fraction D was applied to a 1.6×28-cm column of DEAE-cellulose,equilibrated with the second buffer. The column was washed and thepolymerase was eluted with a linear gradient of 0.01-0.5M potassiumphosphate in the second buffer. The fractions were assayed forcontaminating endonuclease(s) and exonuclease(s) by electrophoreticallydetecting the change in molecular weight of phage λ DNA or supercoiledplasmid DNA after incubation with an excess of DNA polymerase (forendonuclease) and after treatment with a restriction enzyme that cleavesthe DNA into several fragments (for exonuclease). Only those DNApolymerase fractions (65-95 mM potassium phosphate) having minimalnuclease contamination were pooled. To the pool was added autoclavedgelatin in an amount of 250 μg/ml, and dialysis was conducted againstthe second buffer to yield Fraction E.

Fraction E was applied to a phosphocellulose column and eluted with a100 ml gradient (0.01-0.4M KCl gradient in 20 mM potassium phosphatebuffer pH 7.5). The fractions were assayed for contaminatingendo/exonuclease(s) as described above as well as for polymeraseactivity (by the method of Kaledin et al.) and then pooled. The pooledfractions were dialyzed against the second buffer, then concentrated bydialysis against 50% glycerine and the second buffer.

The molecular weight of the polymerase was determined by SDS-PAGEanalysis. Marker proteins (Bio-Rad low molecular weight standards) werephosphorylase B (92,500), bovine serum albumin (66,200), ovalbumin(45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor(21,500), and lysozyme (14,400).

Preliminary data suggest that the polymerase has a molecular weight ofabout 86,000-90,000 daltons, not 62,000-63,000 daltons reported in theliterature (e.g., by Kaledin et al.).

The polymerase was incubated in 50 μl of a mixture containing either 25mM Tris-HCl pH 6.4 or pH 8.0, and 0.1M KCl, 10 mM MgCl₂, 1 mM2-mercaptoethanol, 10 nmoles each of dGTP, dATP, and TTP, and 0.5 μCi (³H) dCTP, 8 μg "activated" calf thymus DNA, and 0.5-5 units of thepolymerase. "Activated" DNA is a native preparation of DNA after partialhydrolysis with DNase I until 5% of the DNA was transferred to theacid-soluble fraction. The reaction was conducted at 70° C. for 30minutes, and stopped by adding 50 μl of a saturated aqueous solution ofsodium pyrophosphate containing 0.125M EDTA-Na₂. Samples were processedand activity was determined as described by Kaledin et al., supra.

The results showed that at pH 6.4 the polymerase was more than one-halfas active as at pH 8.0. In contrast, Kaledin et al. found that at pHabout 7.0, the enzyme therein had 8% of the activity at pH 8.3.Therefore, the pH profile for the thermostable enzyme herein is broaderthan that for the Kaledin et al. enzyme.

Finally, when only one or more nucleotide triphosphates were eliminatedfrom a DNA polymerase assay reaction mixture, very little, if any,activity was observed using the enzyme herein, and the activity wasconsistent with the expected value, and with an enzyme exhibiting highfidelity. In contrast, the activity observed using the Kaledin et al.(supra) enzyme is not consistent with the expected value, and suggestsmisincorporation of nucleotide triphosphate(s).

D. Amplification Reaction

One microgram of the genomic DNA described above was diluted in aninitial 100 μl aqueous reaction volume containing 25 mM Tris.HCl buffer(pH 8.0), 50 mM KCl, 10 mM MgCl₂, 5 mM dithiothreitol, 200 μg/mlgelatin, 1 μM of primer PC03, 1 μM of primer PC04, 1.5 mM dATP, 1.5 mMdCTP, 1.5 mM dGTP and 1.5 mM TTP. The sample was heated for 10 minutesat 98° C. to denature the genomic DNA, then cooled to room temperature.Four microliters of the polymerase from Thermus aquaticus was added tothe reaction mixture and overlaid with a 100 μl mineral oil cap. Thesample was then placed in the aluminum heating block of the liquidhandling and heating instrument described in now abandoned Ser. No.833,368 filed Feb. 25, 1986, the disclosure of which is incorporatedherein by reference.

The DNA sample underwent 20 cycles of amplification in the machine,repeating the following program cycle:

1) heating from 37° C. to 98° C. in heating block over a period of 2.5minutes; and

2) cooling from 98° C. to 37° C. over a period of three minutes to allowthe primers and DNA to anneal.

After the last cycle, the sample was incubated for an additional 10minutes at 55° C. to complete the final extension reaction.

E. Synthesis and Phosphorylation of Oligodeoxyribonucleotide Probes

A labeled DNA probe, designated RS24, of the following sequence wasprepared:

    5'-*CCCACAGGGCAGTAACGGCAGACTTCTCCTCAGGAGTCAG-3'            (RS24)

where * indicates the label. This probe is 40 bases long, spans thefourth through seventeenth condons of the gene, and is complementary tothe normal β-globin allele (β^(A)). The schematic diagram of primers andprobes is given below: ##STR7##

This probe was synthesized according to the procedures described inSection I of Example I. The probe was labeled by contacting 20 pmolethereof with 4 units of T4 polynucleotide kinase (New England Biolabs)and about 40 pmole γ-³² P-ATP (New England Nuclear, about 7000 Ci/mmole)in a 40 μl reaction volume containing 70 mM Tris buffer (pH 7.6), 10 mMMgCl₂, 1.5 mM spermine, and 10 mM dithiothreitol for 60 minutes at 37°C. The total volume was then adjusted to 100 μl with 25 mM EDTA and theprobe purified according to the procedure of Maniatis et al., MolecularCloning (1982), 466-467 over a 1 ml Bio Gel P-4 (BioRad) spin dialysiscolumn equilibrated with Tris-EDTA (TE) buffer (10 mM Tris buffer, 0.1mM EDTA, pH 8.0). TCA precipitation of the reaction product indicatedthat for RS24 the specific activity was 4.3 μCi/pmole and the finalconcentration was 0.118 pmole/μl.

F. Dot Blot Hybridizations

Four microliters of the amplified sample from Section IV and 5.6 μl ofappropriate dilutions of β-globin plasmid DNA calculated to representamplification efficiencies of 70, 75, 80, 85, 90, 95 and 100% werediluted with 200 μl 0.4N NaOH, 25 mM EDTA and spotted onto a Genatran 45(Plasco) nylon filter by first wetting the filter with water, placing itin a Bio-Dot (Bio-Rad, Richmond, Calif.) apparatus for preparing dotblots which holds the filters in place, applying the samples, andrinsing each well with 0.1 ml of 20×SSPE (3.6M NaCl, 200 mM NaH₂ PO₄, 20mM EDTA), as disclosed by Reed and Mann, Nucleic Acids Research, 13,7202-7221 (1985). The filters were then removed, rinsed in 20×SSPE, andbaked for 30 minutes at 80° C. in a vacuum oven.

After baking, each filter was then contacted with 16 ml of ahybridization solution consisting of 3×SSPE, 5×Denhardt's solution(1×=0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serumalbumin, 0.2 mM Tris, 0.2 mM EDTA, pH 8.0), 0.5% SDS and 30% formamide,and incubated for two hours at 42° C. Then 2 pmole of probe RS24 wasadded to the hybridization solution and the filter was incubated for twominutes at 42° C.

Finally, each hybridized filter was washed twice with 100 ml of 2×SSPEand 0.1% SDS for 10 minutes at room temperature. Then the filters weretreated once with 100 ml of 2×SSPE, 0.1% SDS at 60° C. for 10 minutes.

Each filter was then autoradiographed, with the signal readily apparentafter two hours.

G. Discussion of Autoradiogram

The autoradiogram of the dot blots was analyzed after two hours andcompared in intensity to standard serial dilution β-globinreconstructions prepared with HaeIII/MaeI-digested pBR:β^(A), whereβ^(A) is the wild-type allele, as described in Saiki et al., Science,supra. Analysis of the reaction product indicated that the overallamplification efficiency was about 95%, corresponding to a 630,000-foldincrease in the β-globin target sequence.

EXAMPLE II A. Amplification Reaction

Two 1 μg samples of genomic DNA extracted from the Molt 4 cell line asdescribed in Example I were each diluted in a 100 μl reaction volumecontaining 50 mM KCl, 25 mM Tris.HCl buffer pH 8.0, 10 mM MgCl₂, 1 μM ofprimer PC03, 1 μM of primer PC04, 200 μg/ml gelatin, 10%dimethylsulfoxide (by volume), and 1.5 mM each of dATP, dCTP, dGTP andTTP. After this mixture was heated for 10 minutes at 98° C. to denaturethe genomic DNA, the samples were cooled to room temperature and 4 μl ofthe polymerase from Thermus aquaticus described in Example I was addedto each sample. The samples were overlaid with mineral oil to preventcondensation and evaporative loss.

One of the samples was placed in the heating block of the machinedescribed in Example I and subjected to 25 cycles of amplification,repeating the following program cycle:

(1) heating from 37° to 93° C. over a period of 2.5 minutes;

(2) cooling from 93° C. to 37° C. over a period of three minutes toallow the primers and DNA to anneal; and

(3) maintaining at 37° C. for two minutes.

After the last cycle the sample was incubated for an additional 10minutes at 60° C. to complete the final extension reaction.

The second sample was placed in the heat-conducting container of themachine, described in more detail in EP 236,069, supra. Theheat-conducting container is attached to Peltier heat pumps which adjustthe temperature upwards or downwards and a microprocessor controller tocontrol automatically the amplification sequence, the temperaturelevels, the temperature ramping and the timing of the temperature.

The second sample was subjected to 25 cycles of amplification, repeatingthe following program cycle:

(1) heating from 37° to 95° C. over a period of three minutes;

(2) maintaining at 95° C. for 0.5 minutes to allow denaturation tooccur;

(3) cooling from 95° to 37° C. over a period of one minute; and

(4) maintaining at 37° C. for one minute.

B. Analysis

Two tests were done for analysis, a dot blot and an agarose gelanalysis.

For the dot blot analysis, a labeled DNA probe, designated RS18, of thefollowing sequence was prepared.

    5'-*CTCCTGAGGAGAAGTCTGC-3'(RS18)

where * indicates the label. This probe is 19 bases long, spans thefourth through seventeenth codons of the gene, and is complementary tothe normal β-globin allel (β^(A)). The schematic diagram of primers andprobes is given below: ##STR8##

This probe was synthesized according to the procedures described inSection I of Example I. The probe was labeled by contacting 10 pmolethereof with 4 units of T4 polynucleotide kinase (New England Biolabs)and about 40 pmole γ⁻³² P-ATP (New England Nuclear, about 7000 Ci/mmole)in a 40 μl reaction volume containing 70 mM Tris.HCl buffer (pH 7.6), 10mM MgCl₂, 1.5 mM spermine and 10 mM dithiothreitol for 60 minutes at 37°C. The total volume was then adjusted to 100 μl with 25 mM EDTA andpurified according to the procedure of Maniatis et al., supra, p.466-467 over a 1 ml Bio Gel P-4 (BioRad) spin dialysis columnequilibrated with Tris-EDTA (TE) buffer (10 mM Tris.HCl buffer, 0.1 mMEDTA, pH 8.0). TCA precipitation of the reaction product indicated thatfor RS18 the specific activity was 4.6 μCi/pmole and the finalconcentration was 0.114 pmole/μl.

Five microliters of the amplified sample from Section I and of a sampleamplified as described above except using the Klenow fragment of E. coliDNA Polymerase I instead of the thermostable enzyme were diluted with195 μl 0.4N NaOH, 25 mM EDTA and spotted onto two replicated Genatran 45(Plasco) nylon filters by first wetting the filters with water, placingthem in a Bio-Dot (Bio-Rad, Richmond, Calif.) apparatus for preparingdot blots which holds the filters in place, applying the samples, andrinsing each well with 0.4 ml of 20×SSPE (3.6M NaCl, 200 mM NaH₂ PO₄, 20mM EDTA), as disclosed by Reed and Mann, supra. The filters were thenremoved, rinsed in 20×SSPE, and baked for 30 minutes at 80° C. in avacuum oven.

After baking, each filter was then contacted with 6 ml of ahybridization solution consisting of 5×SSPE, 5×Denhardt's solution(1×=0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serumalbumin, 0.2 mM Tris, 0.2 mM EDTA, pH 8.0) and 0.5% SDS, and incubatedfor 60 minutes at 55° C. Then 5 μl of probe RS18 was added to thehybridization solution and the filter was incubated for 60 minutes at55° C.

Finally, each hybridized filter was washed twice with 100 ml of 2×SSPEand 0.1% SDS for 10 minutes at room temperature. Then the filters weretreated twice more with 100 ml of 5×SSPE, 0.1% SDS at 60° C. for 1) oneminute and 2) three minutes, respectively.

Each filter was then autoradiographed, with the signal readily apparentafter 90 minutes.

In the agarose gel analysis, 5 μl of each amplification reaction wasloaded onto 4% NuSieve/0.5% agarose gel in 1×TBE buffer (0.089M Tris,0.089M boric acid, and 2 mM EDTA) and electrophoresed for 60 minutes at100V. After staining with ethidium bromide, DNA was visualized by UVfluorescence.

The results show that the machines used in Example I and this examplewere equally effective in amplifying the DNA, showing discretehigh-intensity 110-base pair bands of similar intensity, correspondingto the desired sequence, as well as a few other discrete bands of muchlower intensity. In contrast, the amplification method as described inExample I of now abandoned Ser. No. 839,331 filed Mar. 13, 1986, supra,which involves reagent transfer after each cycle using the Klenowfragment of E. coli Polymerase I, gave a DNA smear resulting from thenon-specific amplification of many unrelated DNA sequences.

It is expected that similar improvements in amplification and detectionwould be achieved in evaluating HLA-DQ, DR and DP regions.

If in the above experiments the amplification reaction buffer contains 2mM MgCl₂ instead of 10 mM MgCl₂ and 150-200 μM of each nucleotide ratherthan 1.5 mM of each, and if the lower temperature of 37° C. is raised to45°-58° C. during amplification, better specificity and efficiency ofamplification occur. Also, DMSO was found not necessary or preferred foramplification.

EXAMPLE III Amplification and Cloning

For amplification of a 119-base pair fragment on the human β-globingene, a total of 1 microgram each of human genomic DNA isolated from theMolt 4 cell line or from the GM2064 cell line (representing a homozygousdeletion of the β- and δ-hemoglobin region and available from the HumanGenetic Mutant Cell Depository, Camden, N.J.) as described above wasamplified in a 100 μl reaction volume containing 50 mM KCl, 25 mMTris.HCl pH 8, 10 mM MgCl₂, 200 μg/ml gelatin, 5 mM 2-mercaptoethanol,1.5 mM each of dATP, dCTP, TTP, and dGTP, and 1 μM of each of thefollowing primers:

    5'-CTTCTGcagCAACTGTGTTCACTAGC-3' (GH18)

    5'-CACaAgCTTCATCCACGTTCACC-3' (GH19)

where lower case letters denote mismatches from wild-type sequence tocreate restriction enzyme sites. GH18 is a 26-base oligonucleotidecomplementary to the negative strand and contains an internal PstI site.GH19 is a 23-base oligonucleotide complementary to the plus strand andcontains an internal HindIII recognition sequence. These primers wereselected by first screening the regions of the gene for homology to thePstI and HindIII restriction sites. The primers were then prepared asdescribed in Example I.

The above reaction mixtures were heated for 10 minutes at 95° C. andthen cooled to room temperature. A total of 4 μl of the polymerasedescribed in Example I was added to each reaction mixture, and then eachmixture was overlayed with mineral oil. The reaction mixtures weresubjected to 30 cycles of amplification with the following program:

2.5 min. ramp, 37° to 98° C.

3 min. ramp, 98° to 37° C.

2 min. soak, 37° C.

After the last cycle, the reaction mixtures were incubated for 20minutes at 65° C. to complete the final extension. The mineral oil wasextracted with chloroform and the mixtures were stored at -20° C.

A total of 10 μl of the amplified product was digested with 0.5 μgM13mp10 cloning vector, which is publicly available fromBoehringer-Mannheim, in a 50 μl volume containing 50 mM NaCl, 10 mMTris.HCl, pH 7.8, 10 mM MgCl₂, 20 units PstI and 26 units HindIII for 90minutes at 37° C. The reaction was stopped by freezing at -20° C. Thevolume was adjusted to 110 μl with TE buffer and loaded (100 μl) PG,59onto a 1 ml BioGel P-4 spin dialysis column. One 0.1 ml fraction wascollected and ethanol precipitated.

(At this point it was discovered that there was β-globin amplificationproduct in the GM2064 sample. Subsequent experiments traced the sourceof contamination to the primers, either GH18 or GH19. Because no othersource of primers was available, the experiment was continued with theunderstanding that some cloned sequences would be derived from thecontaminating DNA in the primers.)

The ethanol pellet was resuspended in 15 μl water, then adjusted to 20μl volume containing 50 mM Tris.HCl, pH 7.8, 10 mM MgCl₂, 0.5 mM ATP, 10mM dithiothreitol, and 400 units ligase. This mixture was incubated forthree hours at 16° C.

Ten microliters of ligation reaction mixture containing Molt 4 DNA wastransformed into E. coli strain JM103 competent cells, which arepublicly available from BRL in Bethesda, MD. The procedure followed forpreparing the transformed strain is described in Messing, J. (1981)Third Cleveland Symposium on Macromolecules:Recombinant DNA, ed. A.Walton, Elsevier, Amsterdam, 143-163. A total of 651 colorless plaques(and O blue plaques) were obtained. Of these, 119 had a (+)-strandinsert (18%) and 19 had a (-)-strand insert (3%). This is an increase ofalmost 20-fold over the percentage of β-globin positive plaques amongthe primer-positive plaques from the amplification technique usingKlenow fragment of E. coli Polymerase I, where the reaction proceededfor two minutes at 25° C., after which the steps of heating to 100° C.for two minutes, cooling, adding Klenow fragment, and reacting wererepeated nine times. These results confirm the improved specificity ofthe amplification reaction employing the thermostable enzyme herein.

In a later cloning experiment with GM2064 and the contaminated primers,43 out of 510 colorless plaques (8%) had the (+)-strand insert. Thissuggests that approximately one-half of the 119 clones from Molt 4contain the contaminant sequence.

Ten of the (+)-strand clones from Molt 4 were sequenced. Five werenormal wild-type sequence and five had a single C to T mutation in thethird position of the second codon of the gene (CAC to CAT). Four of thecontaminant clones from GM2064 were sequenced and all four were normal.

Restriction site-modified primers may also be used to amplify and cloneand partially sequence the human N-ras oncogene and to clone base pairsegments of the HLA DQ-α, DQ-β and DR-β genes using the above technique.

Again, if the concentrations of MgCl₂ and nucleotides are reduced to 2mM and 150-200 μM, respectively, and the minimum cycling temperature isincreased from 37° C. to 45°-58° C., the specificity and efficiency ofthe amplification reaction can be increased.

EXAMPLE IV Gene Retrieval A. Identification of a DNA Sequence Probe forthe TAQ Polymerase Gene

A specific DNA sequence probe for the Taq pol gene was obtainedfollowing immunological screening of a λgt11 expression library. T.aquaticus DNA was digested to completion with AluI, ligated with EcoRI12-mer linkers (CCGGAATTCCGG, New England Biolabs), digested with EcoRIand ligated with dephosphorylated, EcoRI-digested λgt11 DNA (PromegaBiotech). The ligated DNA was packaged (Gigapack Plus, Stratagene) andtransfected into E. coli K-12 strain Y1090 (provided by R. Young).

The initial library of 2×10⁵ plaques was screened (Young, R. A., and R.W. Davis (1983) Science, 222: 778-782) with a 1: 2000 dilution of arabbit polyclonal antiserum raised to purified Taq polymerase (seeExamples I and XIII). Candidate plaques were replated at limitingdilution and rescreened until homogeneous (˜3 cycles). Phage werepurified from candidate plaques which failed to react with preimmuneserum and reacted with immune serum.

Candidate phage were used to lysogenize E. coli K-12 strain Y1089 (R.Young). Lysogens were screened for the production of an IPTG induciblefusion protein (larger than β-galactosidase) which reacted with the Taqpolymerase antiserum. Solid phase, size-fractionated fusion proteinswere used to affinity purify epitope-specific antibodies from the totalpolyclonal antiserum (Goldstein, L. S. B., et al. (1986) J. Cell Biol.102: 2076-2087).

The "fished", epitope-selected antibodies were used, in turn, in aWestern analysis to identify which λgt11 phage candidates encoded DNAsequences uniquely specific to Taq polymerase. One λgt11 phagecandidate, designated λgt: 1, specifically selected antibodies from thetotal rabbit polyclonal Taq polymerase antiserum which uniquely reactedwith both purified Taq polymerase and crude extract fractions containingTaq polymerase. This phage, λgt: 1, was used for further study.

The ˜115 bp EcoRI-adapted AluI fragment of Thermus aquaticus DNA waslabeled (Maniatis et al., supra) to generate a Taq polymerase-specificprobe. The probe was used in Southern analyses and to screen a T.aquaticus DNA random genomic library.

B. Construction and Screening of a Thermus Aquaticus Random GenomicLibrary

Lambda phage Charon 35 (Wilhelmine, A. M. et al., supra) was annealedand ligated via its cohesive ends, digested to completion with BamHI,and the annealed arms were purified from the "stuffer" fragments bypotassium acetate density gradient ultracentrifugation (Maniatis, etal., supra). T. aquaticus DNA was partially digested with Sau3A and the15-20 kb size fraction purified by sucrose density gradientultracentrifugation. The random genomic library was constructed byligating the target and vector DNA fragments at a 1:1 molar ratio. TheDNA was packaged and transfected into E. coli K-12 strains LE392 orK802. A library of >20,000 initial phage containing >99% recominants wasamplified on E. coli K-12 strain LE392.

The CH35 Taq genomic phage library was screened (Maniatis et al., supra)with the readiolabeled EcoRI insert of λgt11: 1. Specificallyhybridizing candidate phage plaques were purified and further analyzed.One phage, designated Ch35: 4-2, released ≧ four T. aquaticus DNAfragments upon digestion with HindIII (˜8.0, 4.5, 0.8, 0.58 kb)

The four HindIII T. aquaticus DNA fragments were ligated with HindIIIdigested plasmid BSM13⁺ (3.2 kb, Vector Cloning Systems, San Diego) andindividually cloned following transformation of E. coli K-12 strainDG98.

The ˜8.0 kb HindIII DNA fragment from CH35: 4-2was isolated in plasmidpFC82 (11.2 kb), while the 4.5 kb HindIII DNA fragment from CH35: 4-2was isolated in plasmid pFC83 (7.7 kb).

E. coli strain DG98 harboring pFC82 was shown to contain a thermostable,high temperature DNA polymerase activity (Table 1). In addition, thesecells synthesize a new ˜60 kd molecular weight polypeptide which isimmunologically related to Taq DNA polymerase.

The Taq polymerase coding region of the 8.0 kb HindIII DNA fragment wasfurther localized to the lac-promoter proximal 2.68 kb HindIII to Asp718portion of the 8.0 kb HindIII fragment. This region was subcloned toyield plasmid pFC85 (6.0 kb). Upon induction with IPTG, E. coli DG98cells harboring plasmid pFC85 synthesize up to 100-fold morethermostable, Taq polymerase-related activity (Table 1) than theoriginal parent clone (pFC82/DG98). While cells harboring pFC85synthesize a significant amount of a thermostable DNA polymeraseactivity, only a portion of the Taq pol DNA sequence is translated,resulting in the accumulation of a ˜60 kd Taq polymerase-relatedpolypeptide.

                  TABLE 1                                                         ______________________________________                                        Expression of a Thermostable DNA Polymerase Activity in                       E. coli.sup.#                                                                                Units*/ml                                                      Sample           IPTG    + IPTG                                               ______________________________________                                        BSM13/DG98       --      0.02                                                 pFC82/DG98        2.2    2.7                                                  pFC85/DG98       11.9    643.8                                                ______________________________________                                         .sup.# Cells were grown to late log phase (+/- IPTG, 10 mM), harvested,       sonicated, heated at 75° C. for 20 minutes, centrifuged and the        clarified supernatant assayed at 70° C. for DNA polymerase             activity.                                                                     *1 unit = 1 nMole dCTP incorporated in 30 minutes.                       

EXAMPLE V Expression of Taq Polymerase

The thermostable gene of the present invention can be expressed in anyof a variety of bacterial expression vectors including DG141 (ATCC39588) and pP_(L) N_(RBS) ATG, vectors disclosed in U.S. Pat. No.4,711,845, the disclosure of which is incorporated herein by reference.Both of these host vectors are pBR322 derivatives that have either asequence containing a tryptophan promoter-operator and ribosome bindingsite with an operably linked ATG start codon (DG141) or a sequencecontaining the lambda P_(L) promoter and gene N ribosome binding siteoperably linked to an ATG start codon (pP_(L) N_(RBS) ATG). Either oneof these host vectors may be restricted with SacI, and blunt ended withKlenow or Sl nuclease to construct a convenient restriction site forsubsequent insertion of the Taq polymerase gene.

The full-length Taq polymerase gene was constructed from the DNA insertfragments subcloned into plasmids pFC83 and pFC85 as follows. VectorBSM13⁺ (commercially available from Vector Cloning Systems, San Diego,Calif.) was digested at the unique HindIII site, repaired with Klenowand dNTPs, and ligated with T4 DNA ligase to a BglII octanucleotidelinker, 5'-CAGATCTG-3' (New England Biolabs), and transformed into E.coli strain DG98. Plasmids were isolated from Amp^(R) lacZα⁺transformants. One of the clones was digested with BglII and Asp718restriction enzymes, and the large vector fragment purified by gelelectrophoresis.

Next, plasmid pFC83 was digested with BglII and HindIII and the ˜730base pair fragment was isolated. Plasmid pFC85 was digested with HindIIIand Asp718 and the ˜2.68 kb fragment isolated and joined in athree-piece ligation to the ˜730 base pair BglII-HindIII fragment frompFC83 and the BglII-Asp718 vector fragment of BSM13⁺. This ligationmixture was used to transform E. coli strain DG98 (ATCC 39,768 depositedJuly 13, 1984) from which Amp^(R) colonies were selected and an ˜6.58kilobase plasmid (pLSG1) was isolated. Isopropyl-β-D-thiogalactoside(IPTG)-induced DG98 cells harboring pLSG1 synthesized Taq DNA polymeraseindistinguishable in size from the native enzyme isolated from T.aquaticus.

Oligonucleotide-directed mutagenesis (see Zoller and Smith, Nuc. AcidsRes. (1982) 10: 6487-6500) was used to simultaneously 1) introduce anSphI site within codons 3 to 5 of the Taq DNA polymerase gene sequence(see FIG. 1, nt 8-13), 2) increase the A/T content of four of the firstseven condons without effecting a change in the encoded amino acids(within codons 2-7 in FIG. 1), 3) delete 170 nucleotides of the lacZ DNAand T. aquaticus DNA 5' to the DNA polymerase gene initiation codon.

Bacteriophage R408 (Russel, M., et al., Gene, (1986) 45: 333-338) wasused to infect pLSG1/DG98 cells and direct the synthesis of thesingle-stranded DNA (ss) form (plus strand) of pLSG1. Purified pLSG1ssDNA was annealed with purified PvuII-digested BSM13⁺ BglII vectorfragments and the 47-mer mutagenic oligonucleotide DG26(5'-CCCTTGGGCTCAAAAAGTGGAAGCATGCCTCTCATAGCTGTTTCCTG). Followingextension with E. coli DNA polymerase I Klenow fragment, transformationof DG98 cells, and selection of Amp^(R) transformants, the colonies werescreened with 5' ³² P-labeled DG26. Hybridizing candidates were screenedfor loss of the BglII restriction site, deletion of approximately 170base pairs of lacZ:T. aquaticus DNA, and introduction of a unique SphIsite. One candidate, designated pLSG2, was sequenced and shown to encodethe desired sequence. ##STR9##

Oligonucleotide-directed mutagenesis was used to introduce a uniqueBglII site in plasmid pLSG2 immediately following the TGA stop codon forthe Taq polymerase gene (following nucleotide 2499 in FIG. 1). As above,bacteriophage R408 was used to generate the single-stranded (plus) formof plasmid pLSG2. Purified pLSG2 ssDNA was annealed with purifiedPvuII-digested BSM13⁺ BglII vector fragment and the 29-mer mutagenicoligonucleotide SC107 (5'-GCATGGGGTGGTAGATCTCACTCCTTGGC). Followingextension with Klenow fragment (50 mM each dNTP), transformation of DG98cells and selection for Amp^(R) transformants, colonies were screenedwith 5' ³² P-labeled SC107. Hybridizing candidates were screened foracquisition of a unique BglII site. One candidate, designated pSYC1578,was sequenced and shown to contain the desired sequence. ##STR10##

EXAMPLE VI Construction of expression vectors pDG160 and pDG161

The Amp^(R) or Tet^(R) λP_(L) promoter, gene N ribosome binding site,polylinker, BT cry PRE (BT) (positive retroregulatory element, describedin U.S. Pat. No. 4,666,848, issued May 19, 1987), in a ColE1 cop^(ts)vector were constructed from previously described plasmids and theduplex synthetic oligonucleotide linkers DG31 and DG32. The DG31/32duplex linker encodes a 5' HindIII cohesive end followed by SacI, NcoI,KpnI/Asp718, XmaI/SmaI recognition sites and a 3' BamHI cohesive end.##STR11##

A. Construction of Amp^(R) plasmid pDG160

Plasmid pFC54.t, a 5.96 kb plasmid described in U.S. Pat. No. 4,666,848,supra, was digested with HindIII and BamHI and the isolated vectorfragment was ligated with a 5-fold molar excess of nonphosphorylated andannealed DG31/32 duplex. Following ligation, the DNA was digested withXbaI (to inactivate the parent vector IL-2 DNA fragment) and used totransform E. coli K12 strain DG116 to ampicillin resistance. Colonieswere screened for loss of the des-ala-ser¹²⁵ IL-2 mutein sequence andacquisition of the DG31/32 polylinker sequence by restriction enzymedigestion. The polylinker region in one candidate, designated pDG160,was sequenced and shown to encode the desired polylinker DNA sequence.

B. Construction of Tet^(R) plasmid pDG161

Plasmid pAW740CHB (ATCC 67,605), the source of a modified tetracyclineresistance gene wherein the BamHI and HindIII restriction sites wereeliminated, and which contains the λP_(L) promoter, gene N ribosomebinding site, cry PRE in a ColE1 cop^(ts) vector, was digested tocompletion with HindIII and BamHI and the 4.19 kb vector fragmentpurified by agarose gel electrophoresis. The purified vector DNAfragment was ligated with a 5-fold molar excess of nonphosphorylatedannealed DG31/32 duplex. E. coli K12 strain DG116 was transformed with aportion of the DNA, and Tet^(R) colonies screened for presence of 4.2 kbplasmids. Several candidates were further screened by restriction enzymedigestion and the polylinker region sequenced by the Sanger method. Oneof the candidates with the desired sequence was designated pDG161.

EXAMPLE VII A. Construction of an Amp^(R) P_(L) promoter, gene Nribosome binding site, (N_(RBS)) Taq polymerase (832) BT cry PRE,cop^(ts) expression vector

To express the full-length (832 amino acid) mutated Taq polymerasesequence encoded by plasmid pSYC1578 under the control of the λP_(L)promoter and gene N ribosome binding site, we used plasmids pSYC1578 andpFC54.t. Plasmid pSYC1578 was digested with SphI and BglII and theresulting approximate 2.5 kb Taq polymerase gene fragment purified byagarose gel electrophoresis and electroelution. Plasmid pFC54.t wasdigested to completion with HindIII and BamHI and the vector fragmentpurified by agarose gel electrophoresis. The synthetic oligonucleotidesDG27 (5'-AGCTTATGAGAGGCATG) and DG28 (5'-CCTCTCATA) were synthesized andannealed. Purified pFC54.t fragment (0.085 pmoles), purified Taqpolymerase gene fragment (0.25 pmoles) and annealed nonphosphorylatedDG27/28 duplex adaptor (0.43 pmoles) were combined in 30 μl and ligatedat 14° C. A portion of the ligated DNA was heated to 75° C. (15 minutes)to inactivate the DNA ligase in the samples and treated with XbaI tolinearize (inactivate) any IL-2 mutein containing ligation products. Theligated and digested DNA (approximately 100 ng) was used to transform E.coli K12 strain DG116 to ampicillin resistance. Amp^(R) colonies werescreened for the presence of an approximate 8 kb plasmid which yieldedthe expected digestion products with HindIII (621 bp+7,410 bp), EcoRI(3,250 bp+4,781 bp) and SphI (8,031 bp), Asp718 (8,031 bp), BamHI (8,031bp) and PvuII (4,090 bp+3,477 bp+464 bp). Several candidates weresubjected to DNA sequence analysis at the 5' λP_(L) :TaqPol junction andthe 3' TaqPol:BT junction. One of the candidates was also screened withan anti-Taq polymerase antibody for the synthesis of an approximate 90kd immunoreactive antigen. Single colonies were transferred from a 30°C. culture plate to a 41° C. culture plate for two hours. The colonieswere scraped with a toothpick from both the 30° C. and 41° C. plates,boiled in SDS loading buffer, subjected to SDS-PAGE electrophoresis andthe separated proteins transferred to a nitrocellulose membrane. Themembranes were probed with a 1:6,000 dilution of a polyclonal anti-Taqantibody and developed with a goat anti-rabbit HRP conjugate. All of thecandidates tested showed evidence of temperature inducible approximate90 kd Taq polymerase-related protein. One of the several plasmidcandidates which directed the synthesis of Taq polymerase in E. coli andcontained the expected DNA sequence was designated pLSG5.

B. Construction of a Tet^(R) P_(L) promoter, gene N ribosome bindingsite, Taq polymerase (832) BT cry PRE cop^(ts) expression vector

To express the full length (832 amino acid) mutated Taq polymerasesequence encoded by plasmid pSYC1578 under control of the λP_(L)promoter and gene N ribosome binding site in a Tet^(R) vector, we usedplasmids pSYC1578 and pAW740CHB. Plasmid pSYC1578 was digested with SphIand BglII and the resulting approximate 2.5 kb Taq polymerase genefragment was purified by agarose gel electrophoresis and electroelution.Plasmid pAW740CHB was digested to completion with HindIII and BamHI andthe resulting 4.19 kb vector fragment purified by agarose gelelectrophoresis and electroelution. The synthetic oligonucleotides DG27and DG28 (described previously) were annealed. Purified pAW740CHB vectorfragment (0.12 pmoles) was ligated with purified Taq polymerase genefragment (0.24 pmoles) and annealed nonphosphorylated DG27/28 duplexadaptor (0.24 pmoles) in 30 μl at 14° C. A portion of the ligated DNA(100 ng) was used to transform E. coli K12 strain DG116 to tetracyclineresistance. Tet^(R) candidates were screened for the presence of anapproximate 6.7 kb plasmid which yielded the expected digestion productswith HindIII (621 bp+6,074 bp), EcoRI (3,445 bp+3,250 bp), Asp718 (6,695bp), SphI (3,445 bp+3,250 bp), BamHI (6,695 bp) and PvuII (3,477bp+2,754 bp+464 bp). Several candidates were subjected to DNA sequenceanalysis at the 5' λP_(L) :TaqPol junction and the 3' TaqPol:BTjunction. Candidates were also screened by single colony immunoblot asdescribed above for the temperature inducible synthesis of Taqpolymerase. One of the plasmid candidates which directed the synthesisof Taq polymerase in E. coli and contained the expected DNA sequence wasdesignated pLSG6.

EXAMPLE VIII Construction of a Met4 (v3) 829 Amino Acid Form of TaqPolymerase

The predicted fourth codon of native Taq polymerase directs theincorporation of a methionine residue (see pLSG1 and pLSG2 5' sequencesabove). To obtain a further mutated form of the Taq polymerase gene thatwould direct the synthesis of an 829 amino acid primary translationproduct we used plasmids pSYC1578 and pDG161. Plasmid pSYC1578 wasdigested with SphI, treated with E. coli DNA polymerase I Klenowfragment in the presence of dGTP to remove the four-base 3' cohesive endand generate a CTT (leucine, 5th codon) blunt end. Followinginactivation of the DNA polymerase and concentration of the sample, theDNA was digested with BglII and the approximate 2.5 kb Taq polymerasegene fragment purified by agarose gel electrophoresis andelectroelution. Plasmid pDG161 was digested to completion with SacI,repaired with E. coli DNA polymerase I Klenow fragment in the presenceof dGTP to remove the four base 3' cohesive end and generate an ATGterminated duplex blunt end. Following inactivation of the polymerase,the sample was digested with BamHI.

Digested pDG161 (0.146 pmole) and purified Taq polymerase fragment(0.295 pmole) were ligated at 30 μg/ml under sticky end conditionsovernight. The partially ligated DNA sample (BamHI/BglII ends) wasdiluted to 15 μg/ml and ligated for five hours under blunt endconditions. The DNA ligase was inactivated (75° C., 10 minutes) and thesample digested with NcoI to linearize any ligation products containingthe pDG161 polylinker sequence. Sixty nanograms of the ligated anddigested DNA was used to transform E. coli K12 strain DG116 totetracycline resistance. Tet^(R) candidates were screened for thepresence of an approximate 6.7 kb plasmid which yielded the expecteddigestion products when treated with HindIII (612 bp+6,074 bp), EcoRI(3,445 bp+3,241 bp) and SphI (6,686 bp). Colonies were screened as aboveby single colony immunoblot for the temperature inducible synthesis ofan approximate 90 kd Taq polymerase-related polypeptide. One of theplasmids, designated pLSG7, that directed the synthesis of a Taqpolymerase-related polypeptide was subjected to Sanger sequencedetermination at the 5' λP_(L) promoter:Taq polymerase junction and the3' Taq polymerase:BT junction. Analysis of the DNA sequence at the 5'junction confirmed the restriction enzyme analysis (loss of one of theSphI sites and a 612 bp HindIII fragment, slightly smaller than the 621bp HindIII fragment in pLSG6) and indicated the derivation of a plasmidencoding an 829 amino acid form of Taq polymerase.

EXAMPLE IX Construction of Met289 (v289) 544 Amino Acid Form of TaqPolymerase

During purification of native Taq polymerase (Example XIII) we obtainedan altered form of Taq polymerase that catalyzed the template dependentincorporation of dNTP at 70° C. This altered form of Taq polymerase wasimmunologically related to the approximate 90 kd form described inExample XIII but was of lower molecular weight. Based on mobility,relative to BSA and ovalbumin following SDS-PAGE electrophoresis, theapparent molecular weight of this form is approximately 61 kd. Thisaltered form of the enzyme is not present in carefully prepared crudeextracts of Thermus aquaticus cells as determined by SDS-PAGE Westernblot analysis or in situ DNA polymerase activity determination (Spanos,A., and Hubscher, U. (1983) Meth. Enz. 91: 263-277) following SDS-PAGEgel electrophoresis. This form appears to be proteolytic artifact thatmay arise during sample handling. This lower molecular weight form waspurified to homogeneity and subjected to N-terminal sequencedetermination on an ABI automated gas phase sequencer. Comparison of theobtained N-terminal sequence with the predicted amino acid sequence ofthe Taq polymerase gene (see FIG. 1) indicates this shorter form aroseas a result of proteolytic cleavage between glu₂₈₉ and ser₂₉₀.

To obtain a further truncated form of a Taq polymerase gene that woulddirect the synthesis of a 544 amino acid primary translation product weused plasmids pFC54.t, pSYC1578 and the complementary syntheticoligonucleotides DG29 (5'-AGCTTATGTCTCCAAAAGCT) and DG30(5'-AGCTTTTGGAGACATA). Plasmid pFC54.t was digested to completion withHindIII and BamHI. Plasmid pSYC1578 was digested with BstXI and treatedwith E. coli DNA polymerase I Klenow fragment in the presence of all 4dNTPs to remove the 4 nucleotide 3' cohesive end and generate aCTG-terminated duplex blunt end encoding leu₂₉₄ in the Taq polymerasesequence (see pLSG1, nucleotide 880). The DNA sample was digested tocompletion with BglII and the approximate 1.6 kb BstXI (repaired)/BglIITaq DNA fragment was purified by agarose gel electrophoresis andelectroelution. The pFC54.t plasmid digest (0.1 pmole) was ligated withthe Taq polymerase gene fragment (0.3 pmole) and annealednonphosphorylated DG29/DG30 duplex adaptor (0.5 pmole) under stickyligase conditions at 30 μg/ml, 15° C. overnight. The DNA was diluted toapproximately 10 microgram per ml and ligation continued under blunt endconditions. The ligated DNA sample was digested with XbaI to linearize(inactivate) any IL-2 mutein-encoding ligation products. 80 nanograms ofthe ligated and digested DNA was used to transform E. coli K12 strainDG116 to ampicillin resistance. Amp^(R) candidates were screened for thepresence of an approximate 7.17 kb plasmid which yielded the expecteddigestion products with EcoRI (4,781 bp+2,386 bp), PstI (4,138 bp+3,029bp), ApaI (7,167 bp) and HindIII/PstI (3,400 bp+3,029 bp+738 bp). E.coli colonies harboring candidate plasmids were screened as above bysingle colony immunoblot for the temperature-inducible synthesis of anapproximate 61 kd Taq polymerase related polypeptide. In addition,candidate plasmids were subjected to DNA sequence determination at the5' λP_(L) promoter:Taq DNA junction and the 3' Taq DNA:BT cry PREjunction. One of the plasmids encoding the intended DNA sequence anddirecting the synthesis of a temperature-inducible 61 kd Taq polymeraserelated polypeptide was designated pLSG8.

Yet another truncated Taq polymerase gene contained within the ˜2.68 kbHindIII-Asp718 fragment of plasmid pFC85 can be expressed using, forexample, plasmid pP_(L) N_(RBS) ATG, by operably linking theamino-terminal HindIII restriction site encoding the Taq pol gene to anATG initiation codon. The product of this fusion upon expression willyield an ˜70,000-72,000 dalton truncated polymerase.

This specific construction can be made by digesting plasmid pFC85 withHindIII and treating with Klenow fragment in the presence of dATP anddGTP. The resulting fragment is treated further with Sl nuclease toremove any single-stranded extensions and the resulting DNA digestedwith Asp718 and treated with Klenow fragment in the presence of all fourdNTPs. The recovered fragment can be ligated using T4 DNA ligase todephosphorylated plasmid pP_(L) N_(RBS) ATG, which had been digestedwith SacI and treated with Klenow fragment in the presence of dGTP toconstruct an ATG blunt end. This ligation mixture can then be used totransform E. coli DG116 and the transformants screened for production ofTaq polymerase. Expression can be confirmed by Western immunoblotanalysis and activity analysis.

EXAMPLE X Construction of Amp^(R) Trp Promoter Operator, TrpL RibosomeBinding Site, Taq Polymerase (832) BT Cry PRE Cop^(ts) Expression Vector

To substitute the E. coli trp operon promoter/operator and leaderpeptide ribosome binding site, we used plasmids pLSG5 and pFC52. pFC52was the source of the trp promoter, cop^(ts) and ampicillin resistantdeterminants. However, plasmid pCS4, described in U.S. Pat. No.4,711,845, supra, the disclosure of which is incorporated herein byreference, may be used to provide the identical fragment. Plasmid pLSG5was digested to completion with SphI. The SphI was inactivated (70° C.,10 minutes) and the digested DNA was ligated overnight at 15° C. with anexcess of annealed nonphosphorylated DG27/28 duplex adaptor (see above).The T4 DNA ligase was inactivated (70° C., 10 minutes) and the DNAdigested to completion with MluI. The DNA sample was sequentiallyextracted with phenol and ether, ethanol precipitated and finallyresuspended in 10 mM Tris chloride pH 8, 1 mM EDTA. Plasmid pFC52 (orpCS4) was digested to completion with MluI and extracted with phenol,ether and concentrated as above. The DNA sample was digested tocompletion with HindIII and the HindIII inactivated (75° C., 15minutes). The pLSG5 and pFC52 samples were ligated overnight in equalmolar ratio and at 30 μg/ml under sticky end conditions. The T4 ligasewas inactivated (70° C., 10 minutes) and the ligated DNA was digestedwith XbaI to linearize (inactivate) any IL-2 encoding ligation products(from the pFC52 unwanted, 1.65 kb HindIII/MluI DNA fragment). E. coliK12 strain DG116 was transformed to ampicillin resistance with 30nanogram of the ligated DNA. Amp^(R) colonies were screened for thepresence of approximate 7.78 kb plasmids which yielded the expecteddigestion products with EcoRI (4,781 bp+3,002 bp), SphI (7,783 bp),HindIII (7,162 bp+621 bp), ClaI (7,783 bp) and ClaI/MluI (3,905 bp+3,878bp). Candidate colonies were further screened for expression of anapproximate 90 kd Taq polymerase related protein by single colonySDS-PAGE immunoblotting (as above). Plasmids from two of the candidatesshowing the intended properties were transformed into E. coli K12 strainKB2 (ATCC No. 53075).

By Western immunoblot, both plasmids in both hosts were shown to directthe synthesis of an approximate 90 kd Taq polymerase-related polypeptideupon trp limitation. By Comassie staining of SDS-PAGE fractionated wholecell extract proteins, the trp promoter/Taq polymerase plasmids in E.coli K12 strain KB2 direct the accumulation of significantly more Taqpolymerase than in E. coli K12 strain DG116. One of the plasmids wasdesignated pLSG10.

EXAMPLE XI Synthesis of Recombinant Taq DNA Polymerase Activity in E.coli

E. coli K12 (DG116) strains harboring plasmids pDG160, or pLSG5, orpLSG6 were grown at 32° C. in Bonner-Vogel minimal salts mediacontaining 0.5% glucose, 10 μg/ml thiamine, 0.25% (w/v) Difco casaminoacids and ampicillin (100 μg/ml) or tetracycline (10 μg/ml) asappropriate. Cells were grown to A₆₀₀ of about 0.8 and shifted to 37° C.to simultaneously derepress the lambda P_(L) promoter (inactivation ofcI₈₅₇ repressor) and increase the copy number of the ColE1 cop^(ts)plasmid vector. After six-nine hours of growth at 37° C., aliquots ofthe cells were harvested, the cells centrifuged and the pellets storedat -70° C.

Alternatively, E. coli K12 strain KB2 harboring plasmid pLSG10 was grownfor eight hours at 32° C. in Bonner-Vogel minimal salts media containing0.5% glucose, 5 μg/ml tryptophan, 10 μ/ml thiamine, 0.25% Difco casaminoacids and 100 μg/ml ampicillin to an A₆₀₀ of 3.0. Cells were harvestedas above.

Cell pellets were resuspended to about 62.5 A₆₀₀ /ml (˜150-160 μg totalprotein/ml) in 50 mM Tris-Cl, pH 7.5, 1 mM EDTA, 2.4 mM PMSF and 0.5μg/ml leupeptin and lysed by sonication. Aliquots of the sonicatedextracts were subjected to SDS-PAGE and analyzed by Coomassie stainingand Western immunoblotting with rabbit polyclonal anti-Taq polymeraseantibody. In addition, portions of the extracts were assayed in a hightemperature (74° C.) DNA polymerase assay (see Example XIII below).

Western immunoblotting showed significant induction and synthesis of anapproximately 94 kd Taq DNA polymerase related polypeptide in inducedstrains harboring plasmids pLSG5, 6, and 10. Coomassie blue staining ofSDS-PAGE-separated total cell protein revaled the presence of a newpredominant protein at ˜94 kd in these induced strains. Finally, hightemperature activity assays confirmed the significant level ofrecombinant Taq DNA polymerase synthesis in these E. coli strains (seetable, below).

    ______________________________________                                                                     Uninduced (-)                                               Taq Pol           or        Units*/                                Plasmid Host                                                                             Gene     Promoter Induced (+)                                                                             OD.sub.600                             ______________________________________                                        pDG160/DG116                                                                             -        P.sub.L  - or +    <1.0                                   pLSG5/DG116                                                                              +        P.sub.L  -         23                                     pLSG5/DG116                                                                              +        P.sub.L  +         308                                    pLSG6/DG116                                                                              +        P.sub.L  -         5                                      pLSG6/DG116                                                                              +        P.sub.L  +         170                                    pLSG10/KB2 +        Trp      +         300                                    ______________________________________                                         *1 unit = 10 nmole total nucleotide incorporated at 74° C./30          minutes.                                                                 

EXAMPLE XII Purification of Recombinant Taq DNA Polymerase

E. coli strain DG116 harboring plasmid pLSG5 was grown in a 10 Lfermentor. The medium was 10 mM (NH₄)₂ SO₄, 25 mM KH₂ PO₄, 4 mM Na₃Citrate, 400 μM FeCl₃, 28 μM ZnCl₂, 34 μM CoCl₂, 33 μM NaMoO₄, 27 μMCaCl₂, 30 μM CuCl₂, and 32 μM H₃ BO₃. The medium was adjusted to pH 6.5with NaOH, ˜15 mM, and sterilized. The following sterile components wereadded: 20 mg/l thiamine.HCl, 3 mM MgSO₄, 10 g/l glucose and 12.5 mg/lampicillin. The pH was adjusted to 6.8 and held there using NH₄ OH.Glucose was fed to the culture in conjunction with the alkali demand, tomaintain a glucose concentration at 40% of air saturation, by automaticincreases in rpm (350 to 1000) and airflow (2 to 5 l/min). Foaming wascontrolled on demand using polypropylene glycol.

The fermentor was inoculated with cells and grown to A₆₈₀ =5.0 (14.25hours). The temperature was raised to 37° C. to induce synthesis ofrecombinant Taq polymerase and growth continued for five hours to A₆₈₀of 16.5.

Unless otherwise indicated, all purification steps were conducted at 4°C. Twenty grams (wet weight) of induced frozen E. coli K12 strain DG116harboring plasmid pLSG5 was thawed in 3 volumes of 50 mM Tris-Cl, pH7.5, 1 mM EDTA, 3 mM PMSF, 0.64 μg/ml leupeptin and disrupted in aFrench Press at 20,000 psi. The lysate was adjusted to 5.5× cell volumewith additional buffer and sonicated (4×30 seconds) to reduce viscosity(Fraction I). The crude total cell lysate was adjusted to 0.2M (NH₄)₂SO₄ (26.43 g/l) and centrifuged for 15 minutes at 20,000×G. Thesupernatant (Fraction II) was heated to 75° C. (in a 100° C. water bath)and maintained at 72°-75° C. for 15 minutes to denature E. coli hostproteins. The sample was rapidly cooled to 4° C. by swirling in an icewater bath. After 20 minutes at 0° C., the sample was centrifuged at20,000×G for 15 minutes to precipitate the denatured proteins. Thesupernatant (Fraction III) was applied at 4 ml/hr to a 6 mlPhenyl-Sepharose CL-4B (Pharmacia) column equilibrated with 50 mMTris-Cl, pH 7.5, 1 mM EDTA (Buffer A) containing 0.2M (NH₄)₂ SO₄. Thecolumn was sequentially washed with 3-10 column volumes of a) the samebuffer, b) Buffer A, c) Buffer A containing 20% ethylene glycol toremove nucleic acids and non-Taq polymerase proteins. Taq DNA polymeraseactivity was eluted with 60 ml linear gradient of 0-4M urea in Buffer Acontaining 20% ethylene glycol. The active fractions (˜2M urea) werepooled (Fraction IV) and applied at 3 ml/hr to a 12 ml (1.5×6.0 cm)Heparin-Sepharose CL-6B (Pharmacia) column equilibrated in 50 mMTris-Cl, pH 7.5, 0.1 mM EDTA, 0.2% Tween 20 (Buffer B) containing 0.1MKCl. The column was washed with 2 column volumes of Buffer B containing0.15M KCl. The Taq polymerase was eluted with a 120 ml linear gradientof 0.15-0.65M KCl in Buffer B. The Taq polymerase eluted as a singleA₂₈₀ and activity peak at ˜0.29M KCl.

Purified recombinant and native Taq polymerase proteins comigratefollowing electrophoresis on SDS-PAGE and staining with Coomassie blue.The purified Taq polymerase proteins migrate slightly faster thanpurified Phosphorylase B (Pharmacia), consistent with a molecular weightpredicted from the DNA sequence (of pLSG5) of 93,920 daltons.

The peak activity fractions were pooled and a portion subjected toN-terminal amino acid sequence determination on an Applied Biosystemsgas phase sequencer. In contrast to native Taq polymerase which has ablocked amino terminus, the sequence of the purified recombinant Taqpolymerase and the individual cycle yields were consistent with thesequence predicted for the amino terminus of the Taq polymerase proteinencoded by plasmid pLSG5.

The recombinant Taq polymerase encoded by plasmid pLSG5 and purified asdescribed could amplify a human "single copy" sequence. Using a lowtemperature limit of 55° C., extension temperature of 72° C., uppertemperature limit of 94° C. and a 2-2.5 minute cycle time, comparableyields and efficiency were noted for native and recombinant Taqpolymerase using 1-2 units/100 μl PCR.

EXAMPLE XIII Purification

The thermostable polymerase may be purified directly from a culture ofThermus aquaticus following the example disclosed below or,alternatively, from a bacterial culture containing the recombinantlyproduced enzyme with only minor modifications necessary in thepreparation of the crude extract.

After harvesting by centrifugation, 60 grams of cells were resuspendedin 75 ml of a buffer consisting of 50 mM Tris-Cl pH 8, 1 mM EDTA. Cellswere lysed in a French Press at 14,000-16,000 PSI after which 4 volumes(300 ml) of additional Tris-EDTA were added. Buffer A (β-mercaptoethanolto 5 mM and NP-40 and Tween 20 to 0.5% (v/v) each) was added and thesolution was sonicated thoroughly while cooling. The resultanthomogeneous suspension was diluted further with Buffer A such that thefinal volume was 7.5-8 times the starting cell weight; this wasdesignated Fraction I.

The polymerase activity in Fraction I and subsequent fractions wasdetermined in a 50 μl mixture containing 0.025M TAPS-HCl pH 9.4 (20°C.), 0.002M MgCl₂, 0.05M KCl, 1 mM 2-mercaptoethanol, 0.2 mM each dGTP,dATP, TTP, 0.1 mM dCTP [α-³² P, 0.05 Ci/mM], 12.5 μg "activated" salmonsperm DNA and 0.01-0.2 units of the polymerase (diluted in 10 mMTris-HCl, pH 8, 50 mM KCl, 1 mg/ml autoclaved gelatin, 0.5% NP-40, 0.5%Tween 20, and 1 mM 2-mercaptoethanol). One unit corresponds to 10 nmolesof product synthesized in 30 minutes. "Activated" DNA is a nativepreparation of DNA after partial hydrolysis with DNase I until 5% of theDNA was transferred to the acid-soluble fraction. The reaction wasconducted at 74° C. for 10 minutes and then 40 μl was transferred to 1.0ml of 50 μg/ml carrier DNA in 2 mM EDTA at 0° C. An equal volume (1.0ml) of 20% TCA, 2% sodium pyrophosphate was added. After 15-20 minutesat 0° C. the samples were filtered through Whatman GF/C discs andextensively washed with cold 5% TCA-1% pyrophosphate, followed by cold95% ethanol, dried and counted.

Fraction I was centrifuged for two hours at 35,000 rpm in a Beckman TI45 rotor at 2° C. and the collected supernatant was designated FractionII.

The Taq polymerase activity was precipitated with Polymin P (BRL,Gaithersburg, MD) (10%, w/v, adjusted to pH 7.5 and autoclaved) afterthe minimum amount of Polymin P necessary to precipitate 90-95% of theactivity was determined, which amount was generally found to be between0.25% and 0.3% final volume.

An appropriate level of Polymin P was added slowly to Fraction II whilestirring for 15 minutes at 0° C. This solution was centrifuged at 13,000rpm for 20 minutes in a Beckman JA 14 rotor at 2° C. The supernatant wasassayed for activity and the pellet was resuspended in 1/5 volume of0.5×Buffer A (diluted 1:2 with H₂ O). This suspension was recentrifugedand the pellet resuspended in 1/4 volume of Buffer A containing 0.4MKCl. This suspension was homogenized thoroughly and left overnight at 4°C. The homogenate was centrifuged as above and the collected supernatantdesignated Fraction III.

The protein fraction was collected by "precipitation" at 75% saturationof ammonium sulfate, centrifuged (at 27,000 rpm, SW27 rotor, 30 minutes)and the floating pellicle was resuspended in 50 mM Tris-Cl pH 8, 1 mMEDTA. These steps were repeated and the protein suspension was dialyzedextensively with P-cell buffer (20 mM KPO₄ pH 7.5, 0.5 mM EDTA, 5 mMβ-mercaptoethanol, 5% (w/v) glycerol, 0.5% (v/v) NP-40 and Tween 20)containing 80 mM KCl.

The dialysate was transferred to a centrifuge bottle to which was addedany recovered protein from sacks rinsed with the P-cell buffercontaining 80 mM KCl. Centrifugation was performed at 20,000×g and thetime was reduced to 15 minutes. The supernatant was saved and any pelletremaining was washed, extracted with P-cell buffer and 80 mM KCl, andrecentrifuged. The supernatants were then combined to form Fraction IV.

Fraction IV was applied to a 2.2×22-cm column of phosphocellulose,equilibrated with the P-cell buffer containing 80 mM KCl. The column waswashed (2.5-3 column volumes) with the same buffer and the proteineluted using a linear gradient of 80 to 400 mM KCl in P-cell buffer.Fractions containing DNA polymerase activity (˜0.18-0.20M KCl) werepooled and concentrated 3-4 fold on an Amicon stirred cell and YM30membrane. The cell was rinsed with the P-cell buffer without KCl andadded to the fraction concentrate (0.15M KCl adjusted final volume) toform Fraction V.

Fraction V was applied to a 5 ml Heparin Sepharose CL-6B column(Pharmacia) equilibrated with P-cell buffer and 0.15M KCl. The columnwas washed with 0.15M KCl buffer (3-4 column volumes) and the proteineluted with a linear gradient from 0.15 to 0.65M KCl in P-cell buffer. A1:10 dilution into diluent without gelatin was made for SDS-PAGEanalysis and a subsequent 1:20 dilution into diluent with 1 mg/mlgelatin was made for use in enzyme assays. The activity fractions(eluting at ˜0.3M KCl) were assayed on supercoiled DNA template forspecific and non-specific endoncleases/topoisomerase byelectrophoretically detecting the change in molecular weight ofsupercoiled plasmid DNA after incubation with an excess of DNApolymerase. Exonuclease contamination was detected following incubationwith small linear DNA fragments. In peak fractions, an ˜88-92 kd proteinwas found to be the major band. The major pool, designated Fraction VI,had the highest polymerase activity with minimal detectable endonucleaseactivity when this pool was assayed for 30 minutes at 55° C. with ˜3-5polymerase units/600 ng DNA.

Fraction VI was dialyzed against 10 mM KPO₄ pH 7.5, 5 mMβ-mercaptoethanol, 5% glycerol, 0.2% NP-40, and 0.2% Tween 20 (HAbuffer). The dialyzed sample was applied to a 3 ml column ofhydroxyapatite and the enzyme eluted with a linear gradient of 10 to 250mM KPO₄ pH 7.5, HA buffer. DNA polymerase activity began to elute at 75mM KPO₄ with the peak at 100 mM KPO₄. Active peak fractions were assayedat 1:100-1:300 dilution. As in the prior chromatography step, a 1:10dilution in diluent was prepared without gelatin for SDS-PAGE analysis.Fractions with no significant endonuclease or double-strand exonucleasewhen assayed at 55° C. with 5 polymerase units were pooled anddesignated Fraction VII.

Fraction VII was dialyzed against a solution of 25 mM sodium acetate pH5.2, 5% glycerol, 5 mM β-mercaptoethanol, 0.1 mM EDTA, 0.1% NP-40, and0.1% Tween 20, adjusted to pH 5 at room temperature. The dialyzed samplewas applied to a 2 ml DEAE-Tris-Acryl-M (LKB) column pre-equilibratedand subsequently washed with the same buffer. The fraction containingpolymerase activity that did not adhere to the column was pooled andadjusted to 50 mM NaCl in the same buffer to yield Fraction VIII.

Fraction VIII was applied to a 2 ml CM-Tris-Acryl M (LKB) columnequilibrated with the same buffer (25 mM sodium acetate, 50 mM NaCl, 5%glycerol, 0.1 mM EDTA, 0.1% NP-40, and 0.1% Tween 20). The column waswashed with 4-5 column volumes of the same buffer and the enzyme elutedwith a linear gradient from 50 to 400 mM NaCl in sodium acetate buffer.The polymerase activity peak eluted ˜0.15-0.20M NaCl. The polymeraseactivity was assayed at 1:300 to 1:500 dilution with the first dilution1:10 into diluent without gelatin for the SDS-PAGE analysis. An assayacross the activity peak on supercoiled DNA templates for specific andnon-specific endonuclease/topoisomerase using DNA polymerase assay salts(25 mM TAPS-HCl pH 9.4, 2.0 mM MgCl₂ and 50 mM KCl) at 74° C. wasperformed, as well as assays for nucleases on M13 ss DNA and pBR322fragments. Active fractions with no detectable nuclease(s) were pooledand run on a silver stained SDS-PAGE mini gel. The results show a single˜88-92 kd band with a specific activity of ˜200,000 units/mg.

This specific activity is more than an order of magnitude higher thanthat claimed for the previously isolated Taq polymerase and is at leastan order of magnitude higher than that for E. coli polymerase 1.

EXAMPLE XIV

The Taq polymerase purified as described above in Example XIII was foundto be free of any contaminating Taq endonuclease and exonucleaseactivities. In addition, the Taq polymerase is preferably stored instorage buffer containing from about 0.1 to about 0.5% volume/volume ofeach non-ionic polymeric detergent employed. More preferably the storagebuffer consists of 50% (v/v) glycerol, 100 mM KCl, 20 mM Tris-Cl pH 8.0,0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol, 0.5%v/v NP-40, 0.5% v/v Tween 20, and 200 μg/ml gelatin, and is preferablystored at -20° C.

The stored Taq polymerase was diluted in a buffer consisting of 25 mMTris Cl pH 8.0, 20 mM KCl, 1 mM β-mercaptoethanol, 0.5% NP-40, 0.5%Tween-20, and 500 μg/ml gelatin. A reaction buffer was then preparedcontaining 50 mM KCl, 10 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 0.01% (w/v)gelatin, 200 μM each dNTP, 1 μM each of the primers that define a 500base pair target sequence on a control template from bacteriophage λ,and 2.0-2.5 units Taq polymerase/assay in a final volume of 100 μl.Template was added to the reaction buffer, the sample placed in a 0.5 mlpolypropylene tube, and the sample topped with 100 μl of heavy whitemineral oil to prevent evaporation.

At least a 10⁵ -fold amplification was achieved when the followingconditions were employed, using 1 ng of control template (bacteriophageλDNA) where the target sequence represented approximately 1% of thestarting mass of DNA.

First the template mixture was denatured for one minute, 30 seconds at94° C. by placing the tube in a heat bath. Then the tube was placed in aheat bath at 37° C. for two minutes. Then the tube was placed in a heatbath at 72° C. for three minutes, and then in the heat bath at 94° C.for one minute. This cycle was repeated for a total of 25 cycles. At theend of the 25th cycle, the heat denaturation step at 94° C. was omittedand replaced by extending the 72° C. incubation step by an additionalthree minutes. Following termination of the assay, the samples wereallowed to cool to room temperature and analyzed as described inprevious examples.

The template may be optimally amplified with a different concentrationof dNTPs and a different amount of Taq polymerase. Also, the size of thetarget sequence in the DNA sample will directly impact the minimum timerequired for proper extension (72° C. incubation step). An optimizationof the temperature cycling profile should be performed for eachindividual template to be amplified, to obtain maximum efficiency.

EXAMPLE XV

Taq polymerase purified as described above in Example I was formulatedfor storage as described in the previous example, but without thenon-ionic polymeric detergents. When assayed for activity as describedin that example, the enzyme storage mixture was found to be inactive.When the NP-40 and Tween 20 were added to the storage buffer, the fullenzyme activity was restored, indicating that the presence of thenon-ionic detergents is necessary to the stability of the enzymeformulation.

EXAMPLE XVI

Several 1 μg samples of human genomic DNA were subjected to 20-35 cyclesof amplification as described in Example II, with equivalent units ofeither Klenow fragment or Taq polymerase, and analyzed by agarose gelelectrophoresis and Southern blot. The primers used in these reactions,PC03 and PC04, direct the synthesis ˜of a 110-bp segment of the humanbeta-globin gene. The Klenow polymerase amplifications exhibited thesmear of DNA typically observed with this enzyme, the apparent cause ofwhich is the non-specific annealing and extension of primers tounrelated genomic sequences under what were essentially non-stringenthybridization conditions (1×Klenow salts at 37° C.). Nevertheless, bySouthern blot a specific 110-bp beta-globin target fragment was detectedin all lanes. A substantially different electrophoretic pattern was seenin the amplifications done with Taq polymerase where the single majorband is the 110-bp target sequence. This remarkable specificity wasundoubtedly due to the temperature at which the primers were extended.

Although, like Klenow fragment amplifications, the annealing step wasperformed at 37° C., the temperature of Taq-catalyzed reactions had tobe raised to about 70° C. before the enzyme exhibited significantactivity. During this transition from 37° to 70° C., poorly matchedprimer-template hybrids (which formed at 37° C.) disassociated so thatby the time the reaction reached an enzyme-activating temperature, onlyhighly complementary substrate was available for extension. Thisspecificity also results in a greater yield of target sequence thansimilar amplifications done with Klenow fragment because thenon-specific extension products effectively compete for the polymerase,thereby reducing the amount of 110-mer that can be made by the Klenowfragment.

EXAMPLE XVII

Amplification was carried out of a sample containing 1 μg Molt 4 DNA, 50mM KCl, 10 mM Tris pH 8.3, 10 mM MgCl₂, 0.01% gelatin, 1 μM of each ofthe following primers (to amplify a 150 bp region):

    5'-CATGCCTCTTTGCACCATTC-3'(RS79)

    and

    5'-TGGTAGCTGGATTGTAGCTG-3'(RS80)

1.5 mM of each dNTP, and 5.0 units of Taq polymerase per 100 μl reactionvolume. Three additional samples were prepared containing 2.5, 1.3, or0.6 units of Taq polymerase. The amplification was carried out in thetemperature cycling machine described above using the following cycle,for 30 cycles:

from 70° to 98° C. for 1 minute

hold at 98° C. for 1 minute

from 98° C. to 35°, 45° or 55° C. for 1 minute

hold at 35°, 45° or 55° C. for 1 minute

from 35°, 45° or 55° C. to 70° C. for 1 minute

hold at 70° C. for 30 seconds

At 35° C. annealing temperature, the 2.5 units/100 μl Taq enzymedilution gave the best-signal-to noise ratio by agarose gelelectrophoresis over all other Taq polymerase concentrations. At 45° C.,the 5 units/100 μl Taq enzyme gave the best signal-to-noise ratio overthe other concentrations. At 55° C., the 5 units/100 μl Taq enzyme gavethe best signal-to-noise ratio over the other concentrations and overthe 45° C. annealing and improved yield. The Taq polymerase has morespecificity and better yield at 55° C.

In a separate experiment the Molt 4 DNA was 10-fold serially dilutedinto the cell line GM2064 DNA, containing no β- or δ-globin sequences,available from the Human Genetic Mutant Cell Depository, Camden, N.J.,at various concentrations representing varying copies per cell, andamplification was carried out on these samples as described in thisexample at annealing temperatures of 35° C. and 55° C. At 35° C., thebest that can be seen by agarose gel electrophoresis is 1 copy in 50cells. At 55° C., the best that can be seen is 1/5,000 cells (a 100-foldimprovement over the lower temperature), illustrating the importance ofincreased annealing temperature for Taq polymerase specificity underthese conditions.

In a third experiment, DNA from a cell line 368H containing HIV-positiveDNA, available from B. Poiesz, State University of New York, Syracuse,N.Y., was similarly diluted into the DNA from the SCl cell line(deposited with ATCC on Mar. 19, 1985; an EBV-transformed β cell linehomozygous for the sickle cell allele and lacking any HIV sequences) atvarious concentrations representing varying copies per cell, andamplification was carried out as described in this Example at annealingtemperatures of 35° C. and 55° C., using the primers SK38 and SK39,which amplify a 115 bp region of the HIV sequence:

    5'-ATAATCCACCTATCCCAGTAGGAGAAAT-3'(SK38)

    and

    5'-TTTGGTCCTTGTCTTATGTCCAGAATGC-3'(SK39)

The results by agarose gel electrophoresis showed that only theundiluted 368H sample could be detected with the annealing temperatureat 35° C., whereas at least a 10² dilution can be detected with theannealing temperature at 55° C., giving a 100-fold improvement indetection.

The following bacteriophage and bacterial strains were deposited withthe Cetus Master Culture Collection, 1400 Fifty-Third Street,Emeryville, Calif., USA (CMCC) and with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC). Thesedeposits were made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for purposesof Patent Procedure and the Regulations thereunder (Budapest Treaty).This assures maintenance of a viable culture for 30 years from the dateof deposit. The organisms will be made available by ATCC under the termsof the Budapest Treaty, and subject to an agreement between applicantsand ATCC that assures unrestricted availability upon issuance of thepertinent U.S. patent. Availability of the deposited strains is not tobe construed as a license to practice the invention in contravention ofthe rights granted under the authority of any government in accordancewith its patent laws.

    ______________________________________                                        Deposit                                                                       Designation     CMCC No.  ATCC No.  Deposit                                   ______________________________________                                        CH35:Taq #4-2   3125      40336     5/29/87                                   E. coli DG98/   3128      67422     5/29/87                                   pFC83                                                                         E. coli DG98/   3127      67421     5/29/87                                   pFC85                                                                         E. coli DG95 (λ N.sub.7 N.sub.53                                                       2103      39789     8/7/84                                    cI.sub.857 susP.sub.80)/pFC54.t                                               E. coli DG116/pAW740CHB                                                                       3291      67605     1/12/88                                   ______________________________________                                    

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the cell lines deposited,since the deposited embodiment is intended as a single illustration ofone aspect of the invention and any cell lines that are functionallyequivalent are within the scope of this invention. The deposit ofmaterials therein does not constitute an admission that the writtendescription herein contained is inadequate to enable the practice of anyaspect of the invention, including the best mode thereof, nor are thedeposits to be construed as limiting the scope of the claims to thespecific illustrations that they represent. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

What is claimed is:
 1. A recombinant DNA sequence that encodes thethermostable DNA polymerase activity of Thermus aquaticus.
 2. Therecombinant DNA sequence of claim 1 shown in FIG.
 1. 3. A recombinantDNA sequence that encodes amino acid residues 290 through 832 shown inFIG.
 1. 4. The recombinant DNA sequence of claim 3 that encodes aminoacid residues 4 through 832 shown in FIG.
 1. 5. A recombinant DNA vectorthat comprises the DNA sequence of claim 1 and can be used to driveexpression of the thermostable DNA polymerase activity of Thermusaquaticus in a host cell transformed with the vector.
 6. The recombinantvector of claim 5 that is plasmid pLSG1.
 7. The recombinant vector ofclaim 5 that is plasmid pLSG2.
 8. The recombinant vector of claim 5 thatis plasmid pLSG5.
 9. The recombinant vector of claim 5 that is plasmidpLSG6.
 10. The recombinant vector of claim 5 that is plasmid pLSG10. 11.A recombinant DNA vector that comprises the DNA sequence of claim 3 andcan be used to drive expression of a protein that has thermostable DNApolymerase activity in a host cell transformed with the vector.
 12. Therecombinant vector of claim 11 that is plasmid pLSG8.
 13. Therecombinant vector of claim 11 that is plasmid pFC82.
 14. Therecombinant vector of claim 11 that is plasmid pFC85.
 15. A recombinantDNA vector that comprises the DNA sequence of claim 4 and can be used todrive expression of a protein that has thermostable DNA polymeraseactivity in a host cell transformed with the vector.
 16. The recombinantvector of claim 15 that is plasmid pLSG7.
 17. A recombinant vectorselected from the group consisting of plasmid pFC83, phage CH35:Taq#4-2, and plasmid pSYC1578.
 18. A host cell transformed with a vector ofclaim
 5. 19. The host cell of claim 18 that is E. coli/pLSG1.
 20. Thehost cell of claim 18 that is E. coli/pLSG2.
 21. The host cell of claim18 that is E. coli/pLSG5.
 22. The host cell of claim 18 that is E.coli/pLSG6.
 23. The host cell of claim 18 that is E. coli/pLSG7.
 24. Thehost cell of claim 18 that is E. coli/pLSG8.
 25. The host cell of claim18 that is E. coli/pLSG10.
 26. The host cell of claim 18 that is E.coli/pFC82.
 27. The host cell of claim 18 that is E. coli/pFC85.